Aminoacyl trna synthetases for modulating inflammation

ABSTRACT

Inflammatory and other cellular response-modulating compositions are provided comprising aminoacyl-tRNA synthetase polypeptides, including active fragments and/or variants thereof. Also provided are methods of using such compositions in the treatment of conditions that benefit from the modulation of inflammation, such as inflammatory diseases or conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/762,151, filed Feb. 7, 2017, which is a continuation application ofU.S. application Ser. No. 13/514,952, filed Sep. 14, 2012, which is aU.S. national phase application of International Patent Application No.PCT/US2010/059963, filed Dec. 10, 2010, which claims benefit under 35U.S.C. §119(e) to U.S. Provisional Application No. 61/285,913, filedDec. 11, 2009; U.S. Provisional Application No. 61/285,923, filed Dec.11, 2009; and U.S. Provisional Application No. 61/285,919, filed Dec.11, 2009, which are incorporated by reference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ATYR-02003US_ST25_SEQ_LISTING.txt. The text fileis about 121 KB, was created on Nov. 14, 2014, and is being submittedelectronically via EFS-Web.

BACKGROUND

1. Technical Field

The present invention relates generally to compositions comprisingaminoacyl-tRNA synthetase polypeptides, including truncations,proteolytic fragments, and/or variants thereof, and methods of usingsuch compositions for modulating inflammation and other cellularresponses.

2. Description of the Related Art

Aminoacyl-tRNA synthetases, which catalyze the aminoacylation of tRNAmolecules, are essential for decoding genetic information during theprocess of translation. In higher eukaryotes, aminoacyl-tRNA synthetasesassociate with other polypeptides to form supramolecular multienzymecomplexes. Each of the eukaryotic tRNA synthetases consists of a coreenzyme, which is closely related to the prokaryotic counterpart of thetRNA synthetase, and one or more additional domains that are appended tothe amino-terminal or carboxyl-terminal end of the core enzyme. Humantyrosyl-tRNA synthetase (YRS), for example, has a carboxyl-terminaldomain that is not part of prokaryotic and lower eukaryotic YRSmolecules.

Aminoacyl tRNA synthetases, such as tyrosyl-tRNA synthetase,tryptophan-tRNA synthetase, and others, are associated with expandedfunctions in mammalian cells, including activities in signaltransduction pathways, among others.

BRIEF SUMMARY

Embodiments of the present invention stem from the unexpected findingthat compositions comprising aminoacyl-tRNA synthetase (AARS)polypeptides, including truncated fragments, proteolytic fragments, andvariants thereof, modulate inflammatory responses, and thereby modulateinflammation. These AARS polypeptides are therefore useful in treating avariety of inflammatory diseases or conditions.

Accordingly, embodiments of the present invention relate generally to tocompositions for modulating inflammation, comprising one or moreisolated aminoacyl-tRNA synthetase (AARS) polypeptides, or biologicallyactive fragments or variants thereof, wherein the polypeptides modulateinflammation. In certain embodiments, the AARS polypeptide is atyrosyl-tRNA synthetase (YRS), a tryptophanyl-tRNA synthetase (WRS), aglutaminyl-tRNA synthetase (QRS), a glycyl-tRNA synthetase (GlyRS), ahistidyl-tRNA synthetase (HisRS), a seryl-tRNA synthetase, aphenylalanyl-tRNA synthetase, an alanyl-tRNA synthetase, anasparaginyl-tRNA synthetase (AsnRS), an aspartyl-tRNA synthetase(AspRS), a cysteinyl-tRNA synthetase (CysRS), a glutamyl-tRNAsynthetase, a prolyl-tRNA synthetase (ProRS), an arginyl-tRNAsynthetase, an isoleucyl-tRNA synthetase, a leucyl-tRNA synthetase, alysyl-tRNA synthetase, a threonyl-tRNA synthetase, a methionyl-tRNAsynthetases, or a valyl-tRNA synthetase.

Certain embodiments include a proteolytic fragment of the AARSpolypeptide. In certain embodiments, the sequence of the proteolyticfragment is derived by incubating the polypeptide with a protease invitro. In certain embodiments, the sequence of the proteolytic fragmentis derived by recombinantly expressing the AARS polypeptide in a cell,wherein the cell comprises one or more recombinant or endogenousproteases. In certain embodiments, the proteolytic fragment comprisesthe sequence of an endogenous, naturally-occurring human or mouse AARSproteolytic fragment.

In certain embodiments, the aminoacyl-tRNA synthetase is a YRSpolypeptide. In certain embodiments, the YRS polypeptide is truncated atits C-terminus. In certain embodiments, the YRS polypeptide comprisesthe amino acid sequence of SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14,wherein at least about 1-50, 50-100, 100-150, 150-200, or about 200-250amino acid residues are truncated from its C-terminus.

In certain embodiments, the YRS polypeptide is truncated at itsN-terminus. In certain embodiments, the YRS polypeptide comprises theamino acid sequence of SEQ ID NO: 1, 2, 3, 6, 8, 10, 12, or 14, whereinat least about 1-50, 50-100, 50-100, 100-150, 150-200, or about 200-250amino acid residues are truncated from its N-terminus.

In certain embodiments, the YRS polypeptide comprises an amino acidsequence at least 80%, 90%, 95%, 98%, or 100% identical to the aminoacid sequence set forth in SEQ ID NO:2, wherein the alanine at position341 is not substituted with a tyrosine. In certain embodiments, the YRSpolypeptide comprises an amino acid sequence at least 80%, 90%, 95%,98%, or 100% identical to the amino acid sequence set forth in SEQ IDNO: 1, 2, 3, 6, 8, 10, 12, or 14.

In certain embodiments, the aminoacyl-tRNA synthetase is a GlyRSpolypeptide. In certain embodiments, the GlyRS polypeptide is a fragmentof the full length human glycyl-tRNA synthetase sequence set forth inSEQ ID NO:16. In certain embodiments, the fragment comprises amino acidresidues 367-438 of SEQ ID NO:16, or an active variant thereof. Incertain embodiments, the GlyRS polypeptide comprises an amino acidsequence at least 80%, 90%, 95%, 98%, or 100% identical to the aminoacid sequence set forth in SEQ ID NO:16. In certain embodiments, theGlyRS polypeptide comprises amino acid residues 57-685, 214-685,239-685, 311-685, 439-685, 511-658, 214-438, 367-438, 214-420, 214-338,85-127 1-213, 1-61, 85-214, 333-685, 128-685, 265-685, 483-685 or 25-56of SEQ ID NO:16, or an active fragment thereof.

In certain embodiments, the aminoacyl-tRNA synthetase is a QRSpolypeptide. In certain embodiments, the QRS polypeptide comprises anamino acid sequence at least 80%, 90%, 95%, 98%, or 100% identical tothe amino acid sequence set forth in SEQ ID NO:25. In certainembodiments, the QRS polypeptide is truncated at its C-terminus. Incertain embodiments, the QRS polypeptide comprises the amino acidsequence of SEQ ID NO:25, wherein at least about 1-50, 50-100, 50-100,100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,or about 500-550 amino acid residues are truncated from its C-terminus.In certain embodiments, the QRS polypeptide comprises amino acidresidues 1-183, 1-220, 1-249, or 1-200 of SEQ ID NO:25, or any one ormore of SEQ ID NOS:36-103 or 109-115.

In certain embodiments, the aminoacyl-tRNA synthetase is a HisRSpolypeptide. Certain embodiments comprise HisRS splice variantpolypeptide. In certain embodiments, the HisRS polypeptide comprises atleast the WHEP domain of HisRS. In certain embodiments, the HisRSpolypeptide comprises at least the anticodon binding domain of HisRS. Incertain embodiments, the HisRS polypeptide lacks a functionalaminoacylation domain. In certain embodiments, the HisRS polypeptidecomprises at least the WHEP domain of HisRS and the anticodon bindingdomain of HisRS but lacks a functional aminoacylation domain. In certainembodiments, the HisRS polypeptide comprises the sequence set forth inSEQ ID NO:28, 30, or 32. In certain embodiments, the HisRS polypeptidecomprises an amino acid sequence at least 80%, 90%, 95%, 98%, or 100%identical to the amino acid sequence set forth in SEQ ID NO:28, 30, or32. In certain embodiments, the HisRS polypeptide comprises at least 20contiguous amino acid residues of the sequence set forth in SEQ IDNO:28, 30, or 32.

In certain embodiments, the aminoacyl-tRNA synthetase is a WRSpolypeptide. In certain embodiments, the WRS polypeptide comprises anamino acid sequence at least 80%, 90%, 95%, 98%, or 100% identical tothe amino acid sequence set forth in any one or more of SEQ IDNOS:33-35. In certain embodiments, the WRS polypeptide comprises abiologically active fragment of any one or more of SEQ ID NOS:33-35.

In certain embodiments, the AARS polypeptide is an aspartyl-tRNAsynthetase (AspRS). In certain embodiments, the AspRS polypeptidecomprises an amino acid sequence at least 80%, 90%, 95%, 98%, or 100%identical to the amino acid sequence set forth in SEQ ID NO:105. Incertain embodiments, the AspRS polypeptide consists essentially of aminoacids 1-154 of SEQ ID NO:105.

Certain embodiments include pharmaceutical compositions for modulatingan inflammatory response in a subject, comprising an aminoacyl-tRNAsynthetase (AARS) polypeptide as in any one of claims 1-54 and apharmaceutically acceptable carrier.

Certain embodiments include methods of modulating an inflammatoryresponse, comprising contacting a cell with an effective concentrationof an aminoacyl-tRNA synthetase (AARS) polypeptide having aninflammatory response-modulating activity, thereby modulating theinflammatory response.

In certain embodiments, the cell is an immune cell or a vascular cell.In certain embodiments, the immune cell is a granulocyte, lymphocyte,monocyte/macrophage, dendritic cell, or mast cell. In certainembodiments, the granulocyte is a neutrophil, eosinophil, or basophil.In certain embodiments, the lymphocyte is a B-cell, CD8+ T-cell, CD4+T-cell, natural killer cell, or γδ T-cell. In certain embodiments, thevascular cell is a smooth muscle cell, endothelial cells, or fibroblast.

Certain embodiments include contacting the cell in vitro or ex vivo.Certain embodiments include administering the cell to a subject. Certainembodiments include contacting the cell in a subject by directlyadministering the AARS polypeptide to the subject.

Certain embodiments include reducing an acute inflammatory response,reducing a chronic inflammatory response, or both. Certain embodimentsinclude increasing an acute inflammatory response, increasing a chronicinflammatory response, or both. Certain embodiments include modulatingthe activation, inflammatory molecule secretion, proliferation,activity, migration, or adhesion of one or more immune cells or vascularcells. Certain embodiments include modulating the levels or activity ofone or more inflammatory molecules.

In certain embodiments, the one or more inflammatory molecules compriseplasma-derived inflammatory molecules of any one or more of thecomplement system, kinin system, coagulation system, or fibrinolysissystem. In certain embodiments, the one or more inflammatory moleculescomprise cell-derived inflammatory molecules of any one or more oflysosome granules, vasoactive amines, eicosanoids, cytokines,acute-phase proteins, or nitric oxide. In certain embodiments, the oneor more cytokines are selected from the cytokines in Tables J and K.Certain embodiments include modulating the levels or activity of any oneor more of TNF-α, IL-2, MIP-1β, IL-12(p40), KC, MIP-2, or IL-10.

Certain embodiments include modulating an inflammatory response orinflammatory condition associated with one or more tissues, tissuesystems, or organs selected from skin, hair follicles, nervous system,auditory system or balance organs, respiratory system, gastroesophogealtissues, gastrointestinal system, vascular system, liver, gallbladder,lymphatic/immune system, uro-genital system, musculoskeletal system,adipose tissue, mammaries, and endocrine system.

Certain embodiments include treating hypersensitivity selected from typeI hypersensitivity, type II hypersensitivity, type III hypersensitivity,type IV hypersensitivity, immediate hypersensitivity, antibody mediatedhypersensitivity, immune complex mediated hypersensitivity, T-lymphocytemediated hypersensitivity, and delayed type hypersensitivity.

Certain embodiments include treating an auto-inflammatory conditionselected from familial Mediterranean fever, TNF receptor associatedperiodic syndrome (TRAPS), Hyper-IgD syndrome (HIDS), CIAS1-relateddiseases such as Muckle-Wells syndrome, familial cold auto-inflammatorysyndrome, and neonatal onset multisystem inflammatory disease, PAPAsyndrome (pyogenic sterile arthritis, pyoderma gangrenosum, acne), andBlau syndrome.

Certain embodiments include treating inflammation associated with acancer selected from prostate cancer, breast cancer, colon cancer,rectal cancer, lung cancer, ovarian cancer, testicular cancer, stomachcancer, bladder cancer, pancreatic cancer, liver cancer, kidney cancer,brain cancer, melanoma, non-melanoma skin cancer, bone cancer, lymphoma,leukemia, thyroid cancer, endometrial cancer, multiple myeloma, acutemyeloid leukemia, neuroblastoma, glioblastoma, and non-Hodgkin'slymphoma.

Certain embodiments include treating inflammation associated withsystemic inflammatory response syndrome (SIRS). Certain embodimentsinclude treating inflammation associated with cytokine storm. Certainembodiments include treating inflammation associated with any one ormore of granulomatous inflammation, fibrinous inflammation, purulentinflammation, serous inflammation, or ulcerative inflammation. Certainembodiments include treating inflammation associated with one or morewounds. Certain embodiments include treating inflammation associatedwith chronic obstructive pulmonary disorder (COPD).

Certain embodiments include increasing the inflammatory response totreat a primary or secondary immunodeficiency. In certain embodiments,the primary immunodeficiency is a combined T-cell and B-cellimmunodeficiency, antibody deficiency, well-defined syndrome, immunedysregulation disease, phagocyte disorder, innate immunity disorder, ora complement deficiency.

Certain embodiments include modulating an inflammatory conditionassociated with activity one or more immune cells or vascular cells. Incertain embodiments, the immune cell is a granulocyte, lymphocyte,monocyte/macrophage, dendritic cell, or mast cell. In certainembodiments, the granulocyte is a neutrophil, eosinophil, or basophil.In certain embodiments, the lymphocyte is a B-cell, T-cell, naturalkiller cell. In certain embodiments, the vascular cell is a smoothmuscle cell, endothelial cells, or fibroblast. In certain embodiments,the inflammatory condition is a neutrophil-mediated condition, amacrophage-mediated condition, or a lymphocyte-mediated condition.

Certain aspects of the present invention stem from the discovery thatcertain glutaminyl-tRNA synthetase (QRS) polypeptides possessnon-canonical biological activities of therapeutic relevance. Therefore,according to one aspect, the present invention provides isolated QRSpolypeptides having at least one non-canonical biological activity, aswell active fragments and variants thereof which substantially retainsaid non-canonical activity. “Non-canonical” activity,” as used herein,refers generally to an activity possessed by a QRS polypeptide of theinvention that is other than aminoacylation and, more specifically,other than the addition of glutamine onto a tRNA^(Gln) molecule. Asdetailed herein, in certain embodiments, a non-canonical biologicalactivity exhibited by a QRS polypeptide of the invention may include,but is not limited to, modulation of cell proliferation, modulation ofapoptosis, modulation of cell signaling (e.g., via Akt), modulation ofangiogenesis, modulation of cell migration, modulation of cell binding,modulation of cellular metabolism, modulation of cytokine production(e.g., IL-12, TNF-α), and the like.

In certain embodiments, the QRS polypeptide of the invention is acontiguous fragment of a full length mammalian QRS protein. In a morespecific embodiment, the QRS polypeptide is a contiguous fragment of thehuman or mouse QRS protein sequence set forth in SEQ ID NOS:25, 36-103,or 109-115. Illustratively, the fragments may be of essentially anylength, provided they are not full length and further provided theyretain at least one non-canonical biological activity of interest. Incertain illustrative embodiments, a QRS polypeptide of the inventionwill range in size from about 10-50, 10-100, 10-150, 10-200, 10-250,10-300, 10-350, 10-400, 10-450, 10-500, 10-550, 10-600, 10-650, 10-700,or 10-750 amino acids in length. In certain illustrative embodiments, aQRS polypeptide of the invention will range in size from about 20-50,20-100, 20-150, 20-200, 20-250, 20-300, 20-350, 20-400, 20-450, 20-500,20-550, 20-600, 20-650, 20-700, or 20-750 amino acids in length. Inother embodiments, the QRS polypeptide of the invention will range insize from about 50-100, 50-150, 50-200, 50-250, 50-300, 50-350, 50-400,50-450, 50-500, 50-550, 50-600, 50-650, 50-700, or 50-750 amino acids inlength. In other embodiments, the QRS polypeptide of the invention willrange in size from about 100-150, 100-200, 100-250, 100-300, 100-350,100-400, 100-450, 100-500, 100-550, 100-600, 100-650, 100-700, or100-750 amino acids in length. In still other illustrative embodiments,the QRS polypeptide of the invention will range in size from about200-250, 200-300, 200-350, 200-400, 200-450, 200-500, 200-550, 200-600,200-650, 200-700, or 200-750 amino acids in length.

In further embodiments of the invention, a QRS polypeptide comprises anactive variant (i.e., retains at least one non-canonical biologicalactivity of interest) of a fragment of a QRS protein sequence, such asthe human QRS protein sequence set forth in SEQ ID NO:25. In a morespecific embodiment, the active variant is a polypeptide having at least70%, 80%, 90%, 95%, 98% or 99% identity along its length to a human ormouse QRS sequence set forth in SEQ ID NOS: 25, 36-103, or 109-115.

Other embodiments of the invention provide QRS splice variants and pointmutants, whether naturally or non-naturally occurring, that possess oneor more non-canonical activities. Other embodiments of the inventionprovide QRS proteolytic fragments, whether produced endogenously (i.e.,in a cell) or in vitro, that possess one or more non-canonicalactivities. In certain embodiments, the sequence of the proteolyticfragment is identified by incubating the QRS polypeptide with a proteasein vitro. In certain embodiments, the sequence of the proteolyticfragment is identified by recombinantly expressing the QRS polypeptidein a cell, wherein the cell comprises one or more recombinant orendogenous proteases. In certain embodiments, the proteolytic fragmentcomprises the sequence of an endogenous, naturally-occurring human ormouse QRS proteolytic fragment.

In a more specific embodiment of the invention, a QRS polypeptidecomprises a fragment of the human QRS sequence of SEQ ID NO:25,comprising or consisting essentially of amino acid residues 1-183 (Q1),1-220 (Q2), 1-249 (Q3), or 1-200 (Q4), or an active fragment or variantthereof that substantially retains at least one non-canonical biologicalactivity of interest. In certain embodiments, a QRS polypeptide fragmentcomprises or consists essentially of any one of SEQ ID NOS:36-103 or109-115.

According to another aspect of the invention, there are provided fusionproteins comprising at least one QRS polypeptide as described herein anda heterologous fusion partner.

According to another aspect of the invention, there are providedisolated polynucleotides encoding the polypeptides and fusion proteinsas described herein, as well as expression vectors comprising suchpolynucleotides, and host cell comprising such expression vectors.

According to yet another aspect of the invention, there are providedcompositions, e.g., pharmaceutical compositions, comprisingphysiologically acceptable carriers and at least one of the isolatedpolypeptides, fusion proteins, antibodies, isolated polynucleotides,expression vectors, host cells, etc., of the invention, as describedherein.

Also provided by the present invention, in other aspects, are methodsfor modulating a cellular activity by contacting a cell or tissue with acomposition of the invention, as described herein, wherein the cellularactivity to be modulated is selected from the group consisting of cellproliferation, apoptosis, cell signaling, cellular metabolism,angiogenesis, cell migration, cell binding, cytokine production, and thelike.

In other aspects, the present invention provides methods for treating adisease, disorder or other condition in a subject in need thereof byadministering a composition according to the present invention. By wayof illustration, such diseases, disorders or conditions may include, butare not limited to, cancer, inflammatory disease or condition, immunedisease (including autoimmune disease) and/or conditions associated withabnormal angiogenesis.

SEQUENCE LISTING

SEQ ID NO:1 is the full-length amino acid sequence of human tyrosyl-tRNAsynthetase (YRS).

SEQ ID NO:2 is the amino acid sequence of a Y341A variant of full-lengthhuman YRS.

SEQ ID NO:3 is the amino acid sequence of a C-terminally truncated(amino acids 1-364) human YRS.

SEQ ID NO:4 is a polynucleotide sequence that encodes the full-lengthamino acid sequence of human YRS (SEQ ID NO:1).

SEQ ID NO:5 shows the sequence of an eight amino acid tag.

SEQ ID NO:6 is the amino acid sequence of the SP1 human YRS splicevariant.

SEQ ID NO:7 is the polynucleotide sequence that encodes the SP1 humanYRS splice variant (SEQ ID NO:6).

SEQ ID NO:8 is the amino acid sequence of the SP2 human YRS splicevariant.

SEQ ID NO:9 is the polynucleotide sequence that encodes the SP2 humanYRS splice variant (SEQ ID NO:8)

SEQ ID NO:10 is the amino acid sequence of the SP3 human YRS splicevariant.

SEQ ID NO:11 is the polynucleotide sequence that encodes the SP3 humanYRS splice variant (SEQ ID NO:10).

SEQ ID NO:12 is the amino acid sequence of the SP4 human YRS splicevariant.

SEQ ID NO:13 is the polynucleotide sequence that encodes the SP4 humanYRS splice variant (SEQ ID NO:12).

SEQ ID NO:14 is the amino acid sequence of the SP5 human YRS splicevariant.

SEQ ID NO:15 is the polynucleotide sequence that encodes the SP5 humanYRS splice variant (SEQ ID NO:14).

SEQ ID NO:16 is the full length amino acid sequence of human cytoplasmicglycyl-tRNA synthetase (GlyRS).

SEQ ID NO:17 is a nucleic acid sequence encoding the GlyRS polypeptideof SEQ ID NO:16.

SEQ ID NOS:18-24 represent illustrative peptide sequences analyzed indetermining GlyRS fragment boundaries.

SEQ ID NO:25 is the full-length amino acid sequence of humanglutaminyl-tRNA synthetase (QRS).

SEQ ID NOS:26 and 27 represent illustrative peptide sequences analyzedin determining QRS fragment boundaries.

SEQ ID NO:28 is the full-length amino acid sequence of the histidyl-tRNAsynthetase (HisRS) protein (NP_(—)002100.2).

SEQ ID NO:29 is a nucleic acid coding sequence of the HisRS-SV9 splicevariant.

SEQ ID NO:30 is the amino acid sequence of the HisRS-SV9 splice variantpolypeptide encoded by SEQ ID NO:29.

SEQ ID NO:31 is a nucleic acid coding sequence of the HisRS-SV11 splicevariant.

SEQ ID NO:32 is the amino acid sequence of the HisRS-SV11 splice variantpolypeptide encoded by SEQ ID NO:31.

SEQ ID NO:33 is the amino acid sequence of the main isoform of humantryptophanyl-tRNA synthetase (WRS).

SEQ ID NO:34 is the amino acid sequence of a truncated variant (T2) ofhuman WRS.

SEQ ID NO:35 is the amino acid sequence of a truncated variant (Toltrup)of human WRS.

SEQ ID NOS:36-103 represent various endogenous peptide fragments ofhuman QRS.

SEQ ID NO:104 is the amino acid sequence of a human phenylalanyl-tRNAsynthetase (PheRS) splice variant (PheRS_SV1P).

SEQ ID NO:105 is the amino acid sequence of a full-length humanaspartyl-tRNA synthetase (AspRS) polypeptide.

SEQ ID NO:106 is the amino acid sequence of an N-terminal fragment (F1;amino acids 1-471) of human WRS.

SEQ ID NO:107 is the amino acid sequence of a splice variant (mini-WRS;amino acids 48-471) of human WRS.

SEQ ID NO:108 is the amino acid sequence of a fragment (T1; amino acids71-471) of human WRS).

SEQ ID NOS:109-115 are naturally-occurring, endogenous human QRSproteolytic fragments obtained from human Jurkat T-cells.

SEQ ID NO:116 is the full-length amino acid sequence of mouseglutaminyl-tRNA synthetase (mQRS).

SEQ ID NO:117 is a nucleic acid sequence encoding the human QRSpolypeptide of SEQ ID NO:25.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show the effects of tyrosyl-tRNA synthetase polypeptides onneutrophil migration into the lungs. FIG. 1A shows the effects forY341A, and FIG. 1B shows the effects for mini-YRS, as compared todexamethasone treated positive control cells and untreated control cells(see Example 1).

FIGS. 2A-2B show the effects of histidyl-tRNA synthetase polypeptides ongranulocyte migration into the lungs. FIG. 2A shows reduced migration ofneutrophils, and FIG. 2B shows reduced migration of eosinophils (seeExample 1).

FIG. 3 shows that tyrosyl-tRNA synthetase polypeptides stimulatemigration of 293 and CHO cell lines transfected with the CXCR-2 receptor(see Example 2). The left graph in FIG. 24 shows the results for293/CXCR-2 cells, and the right graph in FIG. 24 shows the results forCHO/CXCR-2 cells.

FIG. 4 shows the stimulatory effects of YRS polypeptides onpolymorphonuclear (PMN) cell migration (see Example 3).

FIGS. 5A-5D show the in vivo and in vitro cytokine release in responseto the D1 AspRS polypeptide (amino acids 1-154 of SEQ ID NO:105). FIG.5A shows circulating serum levels of TNF-α and IL-10 in mice injectedintravenously with 10 mg/kg D1. TNF-α is increased at early time pointsbut is rapidly cleared while the anti-inflammatory cytokine, IL-10,shows a prolonged time course. FIG. 5B shows in vivo serum levels forfive cytokines from mice injected with D1. FIG. 5C shows in vitroanalysis of PBMCs stimulated with D1, with an increase in TNF-α at 4hours that is markedly higher than the full length DRS. FIG. 5D showsthat secreted IL-10 levels are significantly increased at 24 hrs afterD1 treatment of PBMCs.

FIG. 6 shows that recombinant HRS-SV9 and HRS-SV11 splice variantpolypeptides enhance IL-2 secretion in activated Jurkat T cells. Cellswere treated with PMA (25 ng/ml) plus ionomycin (250 ng/ml) with orwithout HRS-SV9 or HRS-SV11, and media was analyzed 48 hours later byELISA.

FIG. 7 shows that HRS-SV9 stimulated PBMCs to secrete TNF-α in a dosedependent manner.

FIGS. 8A-8C shows the domain structure and amino acid sequence of QRS(SEQ ID NO:25), and illustrates the SDS-PAGE separation of fragments ofQRS (SEQ ID NOS: 116 and 117) generated by endogenous proteolysis of thefull-length QRS.

FIG. 9 shows the QRS fragments (designated Q1, Q2, Q3, and Q4) that werecloned into an E. coli protein expression vector for over-expression andpurification.

FIG. 10 shows that pretreatment with all four QRS fragments (Q1, Q2, Q3,and Q4) inhibited the amount of TNF-α released from PBMCs uponstimulation with 0.5 EU/ml LPS.

FIG. 11 shows that pretreatment with the Q4 fragment inhibited theamount of TNF-α released from PBMCs upon stimulation with 0.5 EU/ml LPS,after 4 and 24 hours.

FIG. 12 shows that pretreatment with the Q4 fragment of QRS inhibitedthe amount of IL-12(p40) released from PBMCs upon stimulation with LPS.

FIGS. 13A to 13C show the inhibitor effects of AARS polypeptides on themigration of THP-1 cells. FIG. 13A shows the inhibitory effects of HisRSon THP-1 migration to the chemoattractant CCL-23, FIG. 13B shows theinhibitory effects of AspRS on THP-1 migration to the chemoattractantCCL-23, and FIG. 13C shows the inhibitory effects of p43 polypeptide onTHP-1 migration to the chemoattractant CCL-5.

FIG. 14 shows a Protein Topography and Migration Analysis Platform(PROTOMAP) of cytosolic (blue) and conditioned media (red) QRS peptidefractions from macrophages, along with a representation of the QRSpolypeptide sequence; (purple) indicates that the peptide was found inboth cytosolic and conditioned media fractions. See Example 5.

FIGS. 15A-15D show the amino acid sequences of naturally-occurring,endogenous QRS peptides fragments that correspond to the PROTOMAP ofFIG. 14. In these figures, (blue; italicized) corresponds to peptidesdetected in the cytosol, (red; underlined) corresponds to peptidesdetected in the conditioned media, and (purple; italicized andunderlined) corresponds to peptides detected in both samples. FIG. 15Ashows the peptide fragments for band 6 (full-length QRS), FIG. 15B showsthe peptide fragments for band 9 (C-terminal QRS fragment), and FIGS.15C-D show the peptide fragments for bands 19 and 20 (N-terminal QRSfragment).

FIGS. 16A-16C show the amino acid sequences of endogenous QRS peptides(blue; italicized) that were obtained from human Jurkat T-cells treatedwith staurosporine. FIGS. 16A and 16B show the peptides for bands 18 and19, respectively, obtained from Jurkat T-cells treated withStaurospaurine (STS) for 4 hours. FIG. 16C shows the peptides for band18, obtained from Jurkat T-cells treated with STS for 6 hours.

DETAILED DESCRIPTION

The present invention stems from the discovery that aminoacyl-tRNAsynthetases (AARS) and certain polypeptides derived therefrom possessnon-canonical biological activities of therapeutic relevance. Therefore,according to one aspect, the present invention provides isolated AARSpolypeptides having at least one non-canonical biological activity, aswell as active fragments and variants thereof which substantially retainsaid non-canonical activity.

“Non-canonical” activity,” as used herein, refers generally to anactivity possessed by a AARS polypeptide of the invention that is otherthan the addition of an amino acid onto a tRNA molecule. As detailedherein, in certain embodiments, a non-canonical biological activityexhibited by an AARS polypeptide of the invention may include, but isnot limited to, the modulation of inflammatory responses, includingacute and chronic inflammatory responses, systemic inflammatoryresponses, local inflammatory responses, and inflammatory responses atthe cellular level, whether in vivo, ex vivo, or in vitro. Examples ofinflammatory response-modulating activities include, without limitation,modulating the growth, activity, or trafficking of various immune cells,and modulating the production or secretion of various cytokines. Hence,embodiments of the present invention include AARS polypeptides,including truncations, splice variants, proteolytic fragments, andvariants thereof, which modulate inflammation, such as by increasing ordecreasing an inflammatory response, and thereby possess therapeuticallybeneficial activity in the treatment and prophylaxis of diseases orconditions associated with inflammation.

Advantages of the use of AARS polypeptides over other treatmentsinclude, for example, a different mechanism of action than traditionaltreatments, synergism with inflammatory-based signaling, higher potency,and the benefits associated with using a de-immunized molecule. Otheradvantages will be apparent to a person skilled in the art.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of molecular biologyand recombinant DNA techniques within the skill of the art, many ofwhich are described below for the purpose of illustration. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al., Molecular Cloning: A Laboratory Manual (3^(rd) Edition, 2000);DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); OligonucleotideSynthesis: Methods and Applications (P. Herdewijn, ed., 2004); NucleicAcid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic AcidHybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R. I. (2005)Culture of Animal Cells, a Manual of Basic Technique, 5^(th) Ed. HobokenN.J., John Wiley & Sons; B. Perbal, A Practical Guide to MolecularCloning (3^(rd) Edition 2010); Farrell, R., RNA Methodologies: ALaboratory Guide for Isolation and Characterization (3^(rd) Edition2005).

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

The term “biologically active fragment”, as applied to fragments of areference polynucleotide or polypeptide sequence, refers to a fragmentthat has at least about 0.1, 0.5, 1, 2, 5, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96,97, 98, 99, 100, 110, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900,1000% or more of the activity of a reference sequence. Included withinthe scope of the present invention are biologically active fragments ofat least about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40,50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280,300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500 or more contiguousnucleotides or amino acid residues in length, including all integers inbetween, which comprise or encode a inflammatory response-modulatingactivity of a reference amino-acyl tRNA transferase polynucleotide orpolypeptide, such as the exemplary reference polypeptide sequences ofSEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, 14, 16, 25, 28, 30, 32-108, and109-115, or exemplary the reference nucleotide sequences of SEQ ID NOS:4, 7, 9, 11, 13, 15, 17, 19, and 31.

Biologically active fragments also include naturally occurring splicevariants of a reference AARS sequence, as well as proteolytic fragmentsof AARS polypeptides.

“Proteolytic fragments,” or the sequence of proteolytic fragments, canbe identified or derived according to a variety of techniques. Forinstance, as exemplified herein, proteolytic fragments can be identifiedin vitro, such as by incubating AARS polypeptides with selectedproteases, or they can be identified endogenously (i.e., in vivo). Incertain embodiments, endogenous proteolytic fragments can be generatedor identified, for instance, by recombinantly expressing AARSpolypeptides in a selected microorganism or eukaryotic cell that hasbeen either modified to contain one or more selected proteases, or thatnaturally contains one or more proteases that are capable of acting onan AARS polypeptide, and isolating and characterizing the endogenouslyproduced proteolytic fragments therefrom. Examples of such proteolyticfragments include Q1-Q4, as described herein, as well as the proteolyticfragments illustrated in Tables C-I, including variants thereof.

In certain embodiments, naturally-occurring endogenous proteolyticfragments can be generated or identified, for instance, from variouscellular fractions (e.g., cytosolic, membrane, nuclear) and/or growthmedium of various cell-types, including, for example, macrophages suchas RAW macrophages (e.g., RAW 264.7 macrophages; see Example 5),T-cells, including primary T-cells and T-cell lines such as Jurkats, andnatural killer (NK) cells, among others. In certain embodiments,endogenous proteolytic fragments, however generated, can be identifiedby techniques such as mass-spectrometry, or equivalent techniques. Oncean in vitro or endogenously identified proteolytic fragment has beengenerated or identified, then it can be sequenced and cloned into anexpression vector for recombinant production, or produced synthetically.

Representative biologically active fragments generally participate in aninteraction, e.g., an intramolecular or an inter-molecular interaction.An inter-molecular interaction can be a specific binding interaction oran enzymatic interaction. An inter-molecular interaction can be betweenan AARS polypeptide and a target molecule, such as another AARSpolypeptide or a target molecule involved in modulating the process ofinflammation (e.g., cytokine production or secretion, immune cellmigration or recruitment, immune cell response to self or foreignantigens, adhesion). Biologically active fragments of an AARSpolypeptide include polypeptide fragments comprising amino acidsequences with sufficient similarity or identity to, or which arederived from, the amino acid sequences of any of SEQ ID NOS: 1, 2, 3, 6,8, 10, 12, 14, 16, 25, 28, 30, 32-108, or 109-115, includingbiologically active portions thereof, or are encoded by a nucleotidesequences of SEQ ID NOS: 4, 7, 9, 11, 13, 15, 17, 19, or 31.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notcontribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they materiallyaffect the activity or action of the listed elements.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

By “corresponds to” or “corresponding to” is meant (a) a polynucleotidehaving a nucleotide sequence that is substantially identical orcomplementary to all or a portion of a reference polynucleotide sequenceor encoding an amino acid sequence identical to an amino acid sequencein a peptide or protein; or (b) a peptide or polypeptide having an aminoacid sequence that is substantially identical to a sequence of aminoacids in a reference peptide or protein.

By “derivative” is meant a polypeptide that has been derived from thebasic sequence by modification, for example by conjugation or complexingwith other chemical moieties (e.g., pegylation) or by post-translationalmodification techniques as would be understood in the art. The term“derivative” also includes within its scope alterations that have beenmade to a parent sequence including additions or deletions that providefor functionally equivalent molecules.

As used herein, the terms “function” and “functional” and the like referto a biological, enzymatic, or therapeutic function.

By “gene” is meant a unit of inheritance that occupies a specific locuson a chromosome and consists of transcriptional and/or translationalregulatory sequences and/or a coding region and/or non-translatedsequences (i.e., introns, 5′ and 3′ untranslated sequences).

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., 1984, Nucleic Acids Research 12, 387-395), which is incorporatedherein by reference. In this way sequences of a similar or substantiallydifferent length to those cited herein could be compared by insertion ofgaps into the alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

The term “host cell” includes an individual cell or cell culture thatcan be or has been a recipient of any recombinant vector(s) or isolatedpolynucleotide of the invention. Host cells include progeny of a singlehost cell, and the progeny may not necessarily be completely identical(in morphology or in total DNA complement) to the original parent celldue to natural, accidental, or deliberate mutation and/or change. A hostcell includes cells transfected or infected in vivo or in vitro with arecombinant vector or a polynucleotide of the invention. A host cellwhich comprises a recombinant vector of the invention is a recombinanthost cell.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide,” as used herein, includes apolynucleotide that has been purified from the sequences that flank itin its naturally-occurring state, e.g., a DNA fragment which has beenremoved from the sequences that are normally adjacent to the fragment.Alternatively, an “isolated peptide” or an “isolated polypeptide” andthe like, as used herein, includes the in vitro isolation and/orpurification of a peptide or polypeptide molecule from its naturalcellular environment, and from association with other components of thecell; i.e., it is not significantly associated with in vivo substances.

By “obtained from” is meant that a sample such as, for example, apolynucleotide extract or polypeptide extract is isolated from, orderived from, a particular source of the subject. For example, theextract can be obtained from a tissue or a biological fluid isolateddirectly from the subject.

The term “oligonucleotide” as used herein refers to a polymer composedof a multiplicity of nucleotide residues (deoxyribonucleotides orribonucleotides, or related structural variants or synthetic analoguesthereof) linked via phosphodiester bonds (or related structural variantsor synthetic analogues thereof). Thus, while the term “oligonucleotide”typically refers to a nucleotide polymer in which the nucleotideresidues and linkages between them are naturally occurring, it will beunderstood that the term also includes within its scope variousanalogues including, but not restricted to, peptide nucleic acids(PNAs), phosphoramidates, phosphorothioates, methyl phosphonates,2-O-methyl ribonucleic acids, and the like. The exact size of themolecule can vary depending on the particular application. Anoligonucleotide is typically rather short in length, generally fromabout 10 to 30 nucleotide residues, but the term can refer to moleculesof any length, although the term “polynucleotide” or “nucleic acid” istypically used for large oligonucleotides.

The term “operably linked” as used herein means placing a structuralgene under the regulatory control of a promoter, which then controls thetranscription and optionally translation of the gene. In theconstruction of heterologous promoter/structural gene combinations, itis generally preferred to position the genetic sequence or promoter at adistance from the gene transcription start site that is approximatelythe same as the distance between that genetic sequence or promoter andthe gene it controls in its natural setting; i.e., the gene from whichthe genetic sequence or promoter is derived. As is known in the art,some variation in this distance can be accommodated without loss offunction. Similarly, the preferred positioning of a regulatory sequenceelement with respect to a heterologous gene to be placed under itscontrol is defined by the positioning of the element in its naturalsetting; i.e., the genes from which it is derived.

The recitation “polynucleotide” or “nucleic acid” as used hereindesignates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers topolymeric form of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The terms “polynucleotide variant” and “variant” and the like refer topolynucleotides displaying substantial sequence identity with areference AARS polynucleotide sequence or polynucleotides that hybridizeto an AARS reference sequence under stringent conditions that aredefined hereinafter. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletionor substitution of at least one nucleotide. Accordingly, the terms“polynucleotide variant” and “variant” include polynucleotides in whichone or more nucleotides have been added or deleted, or replaced withdifferent nucleotides. In this regard, it is well understood in the artthat certain alterations inclusive of mutations, additions, deletionsand substitutions can be made to a reference polynucleotide whereby thealtered polynucleotide retains the biological function or activity ofthe reference polynucleotide. Polynucleotide variants include, forexample, polynucleotides having at least 50% (and at least 51% to atleast 99% and all integer percentages in between) sequence identity withthe sequence set forth in SEQ ID NO:4, 7, 9, 11, 13, 15, 17, 19, or 31,or portions thereof that encode a biologically active fragment of anAARS polypeptide. The terms “polynucleotide variant” and “variant” alsoinclude naturally occurring allelic variants.

“Polypeptide,” “polypeptide fragment,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues andto variants and synthetic analogues of the same. Thus, these terms applyto amino acid polymers in which one or more amino acid residues aresynthetic non-naturally occurring amino acids, such as a chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally-occurring amino acid polymers.

The term “aminoacyl-tRNA synthetase” (AARS) refers generally to enzymesthat in their natural or wild-type form are capable of catalyzing theesterification of a specific amino acid or its precursor to one of allits compatible cognate tRNAs to form an aminoacyl-tRNA. In this“canonical” activity, aminoacyl-tRNA synthetases catalyse a two-stepreaction: first, they activate their respective amino acid by forming anaminoacyl-adenylate, in which the carboxyl of the amino acid is linkedin to the alpha-phosphate of ATP by displacing pyrophosphate, and then,when the correct tRNA is bound, the aminoacyl group of theaminoacyl-adenylate is transferred to the 2′ or 3′ terminal OH of thetRNA.

Class I aminoacyl-tRNA synthetases typically have two highly conservedsequence motifs. These enzymes aminoacylate at the 2′-OH of an adenosinenucleotide, and are usually monomeric or dimeric. Class IIaminoacyl-tRNA synthetases typically have three highly conservedsequence motifs. These enzymes aminoacylate at the 3′-OH of the sameadenosine, and are usually dimeric or tetrameric. The active sites ofclass II enzymes are mainly made up of a seven-stranded anti-parallelβ-sheet flanked by α-helices. Although phenylalanine-tRNA synthetase isclass II, it aminoacylates at the 2′-OH.

AARS polypeptides include tyrosyl-tRNA synthetases (YRS),tryptophanyl-tRNA synthetases (WRS), glutaminyl-tRNA synthetases (QRS),glycyl-tRNA synthetases (GlyRS), histidyl-tRNA synthetases, seryl-tRNAsynthetases, phenylalanyl-tRNA synthetases, alanyl-tRNA synthetases,asparaginyl-tRNA synthetases (AsnRS), aspartyl-tRNA synthetases (AspRS),cysteinyl-tRNA synthetases (CysRS), glutamyl-tRNA synthetases,prolyl-tRNA synthetases (ProRS), arginyl-tRNA synthetases,isoleucyl-tRNA synthetases, leucyl-tRNA synthetases, lysyl-tRNAsynthetases, threonyl-tRNA synthetases, methionyl-tRNA synthetases, andvalyl-tRNA synthetases. The full-length wild-type sequences of theseAARS polypeptides are known in the art. Also included within the meaningof AARS polypeptides are aminoacyl tRNA synthetase-interactingmultifunctional proteins (AIMPs), including AIMP-1 (or p43), AIMP-2 (orp38), and AIMP-3 (or p18).

The recitations “polypeptides,” “polypeptide fragments,” “truncatedpolypeptides” or “variants thereof” encompass, without limitation,polypeptides having the amino acid sequence that shares at least 50%(and at least 51% to at least 99% and all integer percentages inbetween) sequence identity with a reference AARS sequence, such as theamino acid sequence of a human or mouse AARS polypeptide, includingbiologically active fragments thereof, such as fragments having at leastabout 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200,220, 240, 260, 280, 300, 350, 400, 450, 500, 550, 600, 650, 700 or morecontiguous amino acids of the reference sequences, including allintegers in between. These recitations further encompass natural allelicvariation of AARS polypeptides that may exist and occur from one genusor species to another. Illustrative reference sequences include thoseset forth in any one of SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, 14, 16, 25,28, 30, 32-108, and 109-115.

AARS polypeptides, including truncations, fragments, and/or variantsthereof, encompass polypeptides that exhibit at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%,200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of thespecific biological activity of a reference AARS polypeptide (e.g., aninflammatory response-modulating activity in a subject or in vitro).Merely by way of illustration, AARS-related non-canonical biologicalactivity may be quantified, for example, by measuring the ability of anAARS polypeptide to reduce migration of immune cells such asgranulocytes to a site of inflammation, including the lungs, or bymeasuring the effect of an AARS polypeptide on an immune cells responseto a given antigen, whether self or foreign. In certain embodiments,AARS polypeptides desensitize immune cells such as neutrophils to anantigen, and thereby reduce the recruitment of these cells to sites ofinflammation. In certain embodiments, AARS polypeptides modulateinflammatory response of immune cells, or modulate the levels oractivities of various inflammatory molecules, among others. Suitable invitro models for assaying immune cell are described herein (seeExample 1) and known in the art. AARS polypeptides, includingtruncations and/or variants thereof, having substantially reducedbiological activity relative to a reference AARS polypeptide are thosethat exhibit less than about 25%, 10%, 5% or 1% of the specific activityof a biologically active reference AARS polypeptide (i.e., having anon-canonical activity).

The recitation polypeptide “variant” refers to polypeptides that aredistinguished from a reference polypeptide by the addition, deletion orsubstitution of at least one amino acid residue. In certain embodiments,a polypeptide variant is distinguished from a reference polypeptide byone or more substitutions, which may be conservative ornon-conservative. In certain embodiments, the polypeptide variantcomprises conservative substitutions and, in this regard, it is wellunderstood in the art that some amino acids may be changed to otherswith broadly similar properties without changing the nature of theactivity of the polypeptide. Polypeptide variants also encompasspolypeptides in which one or more amino acids have been added ordeleted, or replaced with different amino acid residues.

The present invention contemplates the use in the methods describedherein of variants of full-length AARS polypeptides (e.g., a full-lengthYRS polypeptide having a Y341A substitution), truncated fragments offull-length AARS polypeptides, splice variants, proteolytic fragments,including endogenous proteolytic fragments, and variants of suchfragments, as well as their related biologically active fragments.Biologically active fragments of an AARS polypeptide include peptidescomprising amino acid sequences sufficiently similar to, or derivedfrom, the amino acid sequences of a (putative) full-length AARSpolypeptide sequence, such as SEQ ID NO:1, or portions thereof, or thepolypeptides of SEQ ID NOS:2, 3, 6, 8, 10, 12, 14, 16, 25, 28, 30,32-108, or 109-115.

Typically, biologically active fragments comprise a domain or motif withat least one activity of an AARS polypeptide and may include one or more(and in some cases all) of the various active domains, and includefragments having an inflammatory response-modulating activity. In somecases, biologically active fragments of an AARS polypeptide have abiological activity (e.g., modulating cytokine secretion, modulatingmigration of immune cells) that is unique to the particular, truncatedfragment, such that the full-length AARS polypeptide may not have thatactivity. In certain cases, the biological activity may be revealed byseparating the biologically active AARS polypeptide fragment from theother full-length AARS polypeptide sequences, or by altering certainresidues (e.g., Y341A of the YRS polypeptide) of the full-length AARSwild-type polypeptide sequence to unmask the biologically activedomains. A biologically active fragment of a truncated AARS polypeptidecan be a polypeptide fragment which is, for example, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 550, 600,650, 700, 750 or more contiguous or non-contiguous (e.g., splicevariants are sometimes non-contiguous) amino acids, including allintegers in between, of the amino acid sequences set forth in any one ofSEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, 14, 16, 25, 28, 30, 32-108, or109-115, or the known amino acid sequences of the various human AARSpolypeptides. In certain embodiments, a biologically active fragmentcomprises an inflammatory response-modulating sequence, domain, ormotif. Suitably, the biologically-active fragment has no less than about1%, 10%, 25%, or 50% of an activity of the biologically active (i.e.,non-canonical biological activity) polypeptide from which it is derived.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence identity”, “percentage of sequenceidentity” and “substantial identity”. A “reference sequence” is at least12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology,” John Wiley & Sons Inc, 1994-1998, Chapter 15.

A “subject,” as used herein, includes any animal that exhibits asymptom, or is at risk for exhibiting a symptom, which can be treatedwith either an AARS polypeptide of the invention, cells (e.g., stemcells) that have been treated ex vivo or in vitro with an AARSpolypeptide, or both. Suitable subjects (patients) include laboratoryanimals (such as mouse, rat, rabbit, or guinea pig), farm animals, anddomestic animals or pets (such as a cat or dog). Non-human primates and,preferably, human patients, are included. Certain embodiments includesubjects that exhibit, or are at risk for exhibiting, an increased orpathological inflammatory response, or an insufficient inflammatoryresponse.

An “effective concentration” of an aminoacyl-tRNA synthetase polypeptiderefers to an amount that is capable of modulating or regulating aninflammatory response or inflammation in any desired way, as compared toa control polypeptide or no polypeptide, whether in a cell in vitro orex vivo, in a tissue, or in a subject. One example of a inflammatoryresponse-modulating activity includes reducing migration of immune cellssuch as granulocytes (e.g., neutrophils, eosinophils) or lymphocytes toselected tissues, such as the lung. Another example includesdesensitizing immune cells to an antigen. A further example of aninflammatory response-modulating activity includes the modulation ofcytokine production. Other examples will be apparent from thedescription provided herein and the understanding in the art.

An “immune cell” includes any cell of the vertebrate immune system,including lymphocytes such as B-cells, killer T-cells (i.e., CD8+T-cells), helper T-cells (i.e., CD4+ T-cells, including T_(h)1 andT_(h)2 cells), natural killer cells, and γδ T-cells, monocytes,macrophages, neutrophils, dendritic cells, mast cells, eosinophils, andbasophils.

A “megakaryocyte” refers generally to a bone marrow cell that isresponsible for the production of blood thrombocytes (i.e., platelets),which are necessary for normal blood clotting. Megakaryocytes typicallyaccount for 1 out of 10,000 bone marrow cells. Megakaryocytes arederived from pluripotent hematopoietic stem cell precursor cells in thebone marrow. Thrombopoietin (TPO) is the primary signal formegakaryocyte production, i.e., TPO is sufficient but not absolutelynecessary for inducing differentiation of progenitor cells in the bonemarrow towards a final megakaryocyte phenotype. Other molecular signalsfor megakaryocyte differentiation include GM-CSF, IL-3, IL-6, IL-11,chemokines (SDF-1; FGF-4), and erythropoietin.

Megakaryocytes are believed to develop through the following lineage:CFU-Me (pluripotential hematopoietic stem cell orhemocytoblast)->megakaryoblast->promegakaryocyte->megakaryocyte. At themegakaryoblast stage, the cell loses its ability to divide, but is stillable to replicate its DNA and continue development, becoming polyploid.Upon maturation, megakaryocytes begin the process of producingplatelets, or thrombocytes. Thrombopoietin plays a role in inducing themegakaryocyte to form small proto-platelet processes, or cytoplasmicinternal membranes for storing platelets prior to release. Upon release,each of these proto-platelet processes can give rise to 2000-5000 newplatelets. Overall, about ⅔ of the newly-released platelets will remainin circulation and about ⅓ will be sequestered by the spleen. Afterreleasing the platelets, the remaining cell nucleus typically crossesthe bone marrow barrier to the blood and is consumed in the lung byalveolar macrophages. Megakaryocytopenia, also referred to asmegakaryophthisis, is a scarcity of megakaryocytes in the bone marrow.

An “erythrocyte” refers to a red blood cell that consists mainly ofhemoglobin, a complex metalloprotein containing heme groups whose ironatoms temporarily link to oxygen molecules (O₂) in the lungs.Erythrocytes are produced by a process called erythropoiesis, in whichthey develop from committed stem cells through reticulocytes to matureerythrocytes in about 7 days and live a total of about 100-120 days.“Polycythemias” (or erythrocytoses) are diseases characterized by asurplus of erythrocytes, in which the increased viscosity of the bloodcan cause a number of symptoms. “Anemias” are diseases characterized bylow oxygen transport capacity of the blood, because of low red cellcount or some abnormality of the red blood cells or the hemoglobin.

A “granulocyte” refers to a white blood cell that is characterized bythe presence of granules in its cytoplasm. Granulocytes are alsoreferred to as polymorphonuclear leukocytes (PMN or PML), because of thevarying shapes of the nuclei. Examples of granulocytes includeneutrophils, eosinophils, and basophils.

A “neutrophil,” or neutrophil granulocyte, refers generally to anabundant type of white blood cells in humans, which, together withbasophils and eosinophils, form part of the polymorphonuclear cellfamily (PMNs). Neutrophils can be readily identified according to theirunique staining characteristics on hematoxylin and eosin (H&E)histological or cytological preparations. Neutrophils are normally foundin the blood stream, but are one of the first group of inflammatorycells to migrate toward inflammation sites during the beginning (i.e.,acute) phase of inflammation, mainly as a result of infection or cancer.Typically, neutrophils first migrate through the blood vessels, and thenthrough interstitial tissues, following chemical signals (e.g.,interleukin-8 (IL-8), interferon-gamma (IFN-gamma), and C5a) thatoriginate at the site of inflammation. “Neutropenia” refers to thepresence of low neutrophil counts, which may result from a congenital(genetic) disorder, or may develop due to other conditions, as in thecase of aplastic anemia or some kinds of leukemia. “Neutrophilia” refersto an abnormally high neutrophil count.

“Eosinophils,” also called eosinophilic leukocytes, refer to leukocytesthat have coarse round granules of uniform size within their cytoplasm,and which typically have a bilobate (two-lobed) nucleus. The cytoplasmicgranules of eosinophils stain red with the dye eosin. Eosinophilsnormally constitute about 1% to about 3% of the peripheral bloodleukocytes, at a count of about 350 to 650 per cubic millimeter.Eosinophil counts in blood often rise above the normal range duringallergic reactions and parasitic infections, such as worms.“Eosinopenia” refers to a form of agranulocytosis in which the number ofeosinophil granulocyte is lower than expected. “Eosinophilia” refers toan abnormally high number of eosinophils in the blood. For example,eosinophilia can be categorized as mild (less than about 1500eosinophils per cubic millimeter), moderate (about 1500 to about 5000per cubic millimeter), or severe (more than about 5000 per cubicmillimeter). In primary eosinophilia, the increased production ofeosinophils is typically due to an abnormality in hematopoietic stemcells, such as in eosinophilic leukemia. In secondary eosinophilia, theincreased production of eosinophils is typically due to a reactiveprocess driven by cytokines.

Basophils, also called basophilic leukocytes, refer to leukocytes thathave coarse bluish-black granules of uniform size within the cytoplasm,and which typically have a bilobate (two-lobed) nucleus. The cytoplasmicgranules of basophils stain with basic dyes. Basophils normallyconstitute about 0.5% to 3% of the peripheral blood leukocytes.Basophils store and release histamine and serotonin, among otherchemicals. Basophils are capable of ingesting foreign particles, andalso produce, store and release heparin, serotonin, and histamine. Therelease of inflammatory chemicals such as heparin and histamine is oftenassociated with asthma and allergies. Basophils are produced continuallyby stem cells in the bone marrow. “Basopenia” refers to a low basophilcount (e.g., less than about 0.01×10⁹ per liter of blood), and“basophilia” refers to a high basophil count (e.g., more than about 10¹⁰per liter of blood).

“Lymphocytes” refer generally to white blood cells of the vertebrateimmune system, and include B-cells, T-cells (e.g., helper T-cells,cytotoxic T-cells, γδ T-cells), and natural killer (NK) cells.Generally, and merely for illustrative purposes, B-cells produce andsecrete antibodies, T-helper cells release cytokines and growth factorsthat regulate other immune cells, cytotoxic T-cells (CTLs) lyse virallyinfected cells, tumour cells and allografts, and NK cells lyse virallyinfected cells and tumour cells. “Lymphocytopenia” is characterized byabnormally low level of lymphocytes in the blood. The normal totallymphocyte count is typically about 1000 to 4800/μL in adults, and about3000 to 9500/μl, L in children younger than 2 years. At age 6, the lowerlimit of normal total lymphocyte count is about 1500/μl, L.Lymphocytopenia is often characterized by a total lymphocyte count of<1000/μl, L in adults or <3000/μl, L in children younger than 2 years.Specific examples of lymphocytopenia include T-lymphocytopenia, in whichthere are too few T-cells (e.g., CD4+ T-cell counts below about 300cells/μL) but often normal numbers of other lymphocytes, Blymphocytopenia, in which there are too few B lymphocytes but oftennormal numbers of other lymphocytes, and NK lymphocytopenia, in whichthere are there are too few natural killer cells but often normalnumbers of other lymphocytes.

“Lymphocytosis” refers to an abnormally high lymphocyte count, oftencharacterized by a total lymphocyte count that is more than 40% abovenormal. In adults, absolute lymphocytosis is typically present when theabsolute lymphocyte count is greater than 4000 per microliter, in olderchildren greater than 7000 per microliter, and in infants greater than9000 per microliter. Relative lymphocytosis may occur when there is ahigher proportion (greater than 40%) of lymphocytes among the whiteblood cells, and when lymphocyte count (ALC) is normal (less than about4000 per microliter).

The term “modulating” includes “increasing” or “stimulating,” as well as“decreasing” or “reducing,” typically in a statistically significant ora physiologically significant amount.

The terms “enhance” or “enhancing,” or “increase” or “increasing,” or“stimulate” or “stimulating,” refer generally to the ability of one oragents or compositions to produce or cause a greater physiologicalresponse (i.e., downstream effects) in a cell, as compared to theresponse caused by either no AARS polypeptide or a controlmolecule/composition. A measurable physiological response may includegreater cell growth, expansion, adhesion, or migration, among othersapparent from the understanding in the art and the description herein.Among other methods known in the art, in vitro colony formation assaysrepresent one way to measure cellular responses to agents providedherein. A measurable physiological response may also include a clinicalresponse, such as altered inflammation, as measured, for example, bybody temperature, redness, swelling, or other clinical marker ofinflammation. An “increased” or “enhanced” amount is typically a“statistically significant” amount, and may include an increase that is1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g.,500, 1000 times) (including all integers and decimal points in betweenand above 1), e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by noAARS polypeptide (the absence of an agent) or a control composition.

The term “reduce” may relate generally to the ability of one or moreAARS polypeptides of the invention to “decrease” a relevantphysiological or cellular response, such as a symptom of a disease orcondition described herein, as measured according to routine techniquesin the diagnostic art. Examples include decreased migration of immunecells such as granulocytes to the lung, and decreased inflammation ofthe lung. A measurable physiological response may include decreasedinflammation, as measured, for example, by body temperature, redness,swelling, or other clinical marker of inflammation. Relevantphysiological or cellular responses (in vivo or in vitro) will beapparent to persons skilled in the art. A “decrease” in a response maybe statistically significant as compared to the response produced by noAARS polypeptide or a control composition, and may include, for example,a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers inbetween.

“Migration” refers to cellular migration, a process that can be measuredaccording to routine in vitro assays, as described herein and known inthe art (see, e.g., Example 8). Migration also refers to in vivomigration, such as the migration of cells from one tissue to anothertissue (e.g., from bone marrow to peripheral blood, or from peripheralblood to lung tissue), or from a site within one tissue to another sitewithin the same tissue. Migration in vivo (e.g., chemotaxis) oftenoccurs in a response to infection or damaged/irritated tissue.

“Differentiation” refers to the process by which a less specialized(e.g., pluripotent, totipotent, multipotent, etc.) cell becomes a morespecialized cell type.

“Treatment” or “treating,” as used herein, includes any desirable effecton the symptoms or pathology of a disease or condition associated withthe modulation of inflammation, or on the outcome of other primarytreatments (e.g., infections, allergies) that may benefit from themodulation of inflammation, and may include even minimal changes orimprovements in one or more measurable markers of the disease orcondition being treated. “Treatment” or “treating” does not necessarilyindicate complete eradication or cure of the disease or condition, orassociated symptoms thereof. The subject receiving this treatment is anyanimal in need, including primates, in particular humans, and othermammals such as equines, cattle, swine and sheep; and poultry and petsin general. Also included are “prophylactic” treatments, which reducethe risk of developing a relevant disease or condition, or of developingsymptoms associated with the disease or condition. Exemplary markers ofclinical improvement include without limitation altered bodytemperature, alterations in immune cell count, and alterations inbacterial counts, whether following administration of an AARSpolypeptide, following administration of cells that have been treated exvivo or in vitro with an AARS polypeptide, or both.

By “vector” is meant a polynucleotide molecule, preferably a DNAmolecule derived, for example, from a plasmid, bacteriophage, yeast orvirus, into which a polynucleotide can be inserted or cloned. A vectorpreferably contains one or more unique restriction sites and can becapable of autonomous replication in a defined host cell including atarget cell or tissue or a progenitor cell or tissue thereof, or beintegrable with the genome of the defined host such that the clonedsequence is reproducible. Accordingly, the vector can be an autonomouslyreplicating vector, i.e., a vector that exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a linear or closed circular plasmid, anextra-chromosomal element, a mini-chromosome, or an artificialchromosome. The vector can contain any means for assuringself-replication. Alternatively, the vector can be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. A vector system can comprise a single vector or plasmid, twoor more vectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon. The choiceof the vector will typically depend on the compatibility of the vectorwith the host cell into which the vector is to be introduced. In thepresent case, the vector is preferably one which is operably functionalin a bacterial cell. The vector can also include a selection marker suchas an antibiotic resistance gene that can be used for selection ofsuitable transformants.

The terms “wild-type” and “naturally occurring” are used interchangeablyto refer to a gene or gene product that has the characteristics of thatgene or gene product when isolated from a naturally occurring source. Awild-type gene or gene product (e.g., a polypeptide) is that which ismost frequently observed in a population and is thus arbitrarilydesigned the “normal” or “wild-type” form of the gene.

Aminoacyl-tRNA Polypeptides and Variants Thereof

The present invention relates in part to the observation thataminoacyl-tRNA synthetase polypeptides, including truncations andvariants thereof, modulate inflammatory responses both in vivo and exvivo (or in vitro). Accordingly, polypeptides of the present inventioninclude a full-length aminoacyl-tRNA synthetase polypeptide, in additionto any biologically active fragments, or variants or modificationsthereof, of an aminoacyl-tRNA synthetase polypeptide, wherein thepolypeptide is capable of modulating an inflammatory response, either ina subject, in vitro, or ex vivo.

Aminoacyl-tRNA synthetases typically catalyze the aminoacylation of tRNAwith their cognate amino acid. Because of their central role in linkingamino acids with nucleotide triplets contained in tRNAs, aminoacyl-tRNAsynthetases are thought to be among the first proteins that appeared inevolution.

As noted above, examples of aminoacyl-tRNA synthetases includetyrosyl-tRNA synthetases (YRS), tryptophanyl-tRNA synthetases (WRS),glutaminyl-tRNA synthetases (QRS), glycyl-tRNA synthetases (GlyRS),histidyl-tRNA synthetases (HisRS), seryl-tRNA synthetases (SRS),phenylalanyl-tRNA synthetases (PheRS), alanyl-tRNA synthetases (AlaRS),asparaginyl-tRNA synthetases (AsnRS), aspartyl-tRNA synthetases (AspRS),cysteinyl-tRNA synthetases (CysRS), glutamyl-tRNA synthetases (ERS),prolyl-tRNA synthetases (ProRS), arginyl-tRNA synthetases (RRS),isoleucyl-tRNA synthetases (IRS), leucyl-tRNA synthetases (LRS),lysyl-tRNA synthetases (KRS), threonyl-tRNA synthetases (TRS),methionyl-tRNA synthetases (MRS), and valyl-tRNA synthetases (VRS).

Tyrosyl-tRNA synthetases (YRS) belong to the class I tRNA synthetasefamily, which has two highly conserved sequence motifs at the activesite, HIGH and KMSKS. Class I tRNA synthetases aminoacylate at the 2′-OHof an adenosine nucleotide, and are usually monomeric or dimeric (one ortwo subunits, respectively).

The human tyrosyl-tRNA synthetase is composed of three domains: 1) anamino-terminal Rossmann fold domain that is responsible for formation ofthe activated E.Tyr-AMP intermediate and is conserved among bacteria,archeae, and eukaryotes; 2) a tRNA anticodon recognition domain that hasnot been conserved between bacteria and eukaryotes; and 3) acarboxyl-terminal domain that is unique to the human tyrosyl-tRNAsynthetase, and whose primary structure is 49% identical to the putativehuman cytokine endothelial monocyte-activating protein II, 50% identicalto the carboxyl-terminal domain of methionyl-tRNA synthetase fromCaenorhabditis elegans, and 43% identical to the carboxyl-terminaldomain of Arc 1p from Saccharomyces cerevisiae.

The first two domains of the human tyrosyl-tRNA synthetase are 52, 36,and 16% identical to tyrosyl-tRNA synthetases from S. cerevisiae,Methanococcus jannaschii, and Bacillus stearothermophilus, respectively.Nine of fifteen amino acids known to be involved in the formation of thetyrosyl-adenylate complex in B. stearothermophilus are conserved acrossall of the organisms, whereas amino acids involved in the recognition oftRNA^(Tyr) are not conserved. Kinetic analyses of recombinant human andB. stearothermophilus tyrosyl-tRNA synthetases expressed in Escherichiacoli indicate that human tyrosyl-tRNA synthetase aminoacylates human butnot B. stearothermophilus tRNA^(Tyr), and vice versa. It is believedthat the carboxyl-terminal domain of human tyrosyl-tRNA synthetaseevolved from gene duplication of the carboxyl-terminal domain ofmethionyl-tRNA synthetase and may direct tRNA to the active site of theenzyme.

Biological fragments of eukaryotic tyrosyl-tRNA synthetases connectprotein synthesis to cell-signaling pathways. These fragments may beproduced naturally by either alternative splicing or proteolysis, or byartificial proteolytic treatment. For example, as provided in thepresent invention, the N-terminal fragment mini-YRS is capable ofmodulating inflammatory responses in vivo. In addition, certainmutations in the full-length YRS polypeptide sequence confer increasedinflammatory response-modulating activity on the reference sequence(e.g., Y341A). Examples of truncated splice variants of the full-lengthYRS polypeptide sequence include the SP1-SP5 polypeptides.

The full-length amino acid sequence of human tyrosyl-tRNA synthetase isset forth in SEQ ID NO:1. The structure of human mini-YRS (i.e., SEQ IDNO:3; or mini-Tyr), which contains both the catalytic and the anticodonrecognition domain, has been reported to a resolution of 1.18 Å. Whereasthe catalytic domains of the human and bacterial enzymes superimpose,the spatial disposition of the anticodon recognition domain relative tothe catalytic domain is unique in mini-YRS relative to the bacterialorthologs. Without wishing to be bound by any one theory, the uniqueorientation of the anticodon-recognition domain may explain why thefragment mini-YRS is more active in various cell-signaling pathways.

Specific examples of YRS polypeptide variants include full-length YRSpolypeptides, or truncations or splice variants thereof, having one ormore amino acid substitutions selected from an R93Q substitution, anI14L substitution, an N17G substitution, an L271 substitution, an A85Ssubstitution, and a V156L substitution, in addition to combinationsthereof. Particular examples of YRS polypeptide variants include, butare not limited to, a YRS polypeptide having amino acids 1-364 of SEQ IDNO:1 with an R93Q substitution, a YRS polypeptide having amino acids1-353 of SEQ ID NO:1 with an I14L substitution, a YRS polypeptide havingamino acids 1-353 of SEQ ID NO:1 with an N17G substitution, a YRSpolypeptide having amino acids 1-353 of SEQ ID NO:1 with an L27Isubstitution, a YRS polypeptide having amino acids 1-353 of SEQ ID NO:1with an A85S substitution, and a YRS polypeptide having amino acids1-353 of SEQ ID NO:1 with a V156L substitution.

Particular examples of biologically active YRS fragments include, butare not limited to, C-terminally truncated tyrosyl-tRNA synthetasepolypeptides comprising or consisting of amino acids 1-343, amino acids1-344, amino acids 1-350, amino acids 1-353, or amino acids 1-364 of theamino acid sequence set forth in SEQ ID NO:1, in addition to thepolypeptides of SEQ ID NOS:3 and 6. Additional examples of biologicallyactive fragments include, but are not limited to, N-terminally truncatedtyrosyl-tRNA synthetase polypeptides comprising or consisting of theamino acid sequences set forth in SEQ ID NOS: 6, 10, 12, and 14. Theseand other YRS polypeptides are included within the AARS polypeptides ofthe present invention.

Histidyl-tRNA synthetases (HRS or HisRS) are α2 dimers that belong tothe class IIa tRNA synthetase family. A compilation of primarystructures of HisRSs shows that the subunits of these homo-dimericenzymes consist of 420-550 amino acid residues. This represents arelatively short chain length among AARSs, whose peptide chain sizesrange from about 300 to 1100 amino acid residues. SEQ ID NO:28 is theamino acid sequence of the full length HisRS protein (NP_(—)002100.2).SEQ ID NO:30 is the amino acid sequence of the HRS-SV9 splice variant,and SEQ ID NO:32 is the amino acid sequence of the HRS-SV11 splicevariant.

Examples of histidyl-tRNA synthetase polypeptides, and variants ortruncations thereof, include HisRS fragments comprising at least theWHEP domain of HisRS, e.g., amino acid residues 3-43 of the human fulllength HisRS protein and HisRS fragments comprising at least theanticodon binding domain of HisRS, e.g., amino acid residues 406-501 ofthe full length human HisRS protein. Further examples include HisRSfragments that lack a functional aminoacylation domain, e.g., amino acidresidues 54-398 of the human full length HisRS protein or HisRS splicevariant polypeptides that comprise at least the WHEP domain and theanticodon binding domain but lack a functional aminoacylation domain.

In certain embodiments, the HisRS polypeptide of the invention comprisesa sequence set forth in SEQ ID NOS:28, 30, or 32, or is a contiguous ornon-contiguous (e.g., splice variants may be non-contiguous) fragment ofa polypeptide set forth in SEQ ID NOS:28, 30, or 32. Illustratively, thefragments may be of essentially any length, provided they retain atleast one non-canonical biological activity of interest. For example, asfurther described herein, such a fragment may comprise at least about 5,10, 15, 20, 25, 50, 75 or 80, or more, contiguous amino acid residues ofSEQ ID NOS:28, 30, or 32.

In further embodiments of the invention, a HisRS polypeptide comprisesan active variant (i.e., retains at least one non-canonical biologicalactivity of interest) of a sequence set forth in SEQ ID NOS:28, 30, or32. In certain embodiments, the active variant is a polypeptide havingat least 70%, 80%, 90%, 95% or 99% identity along its length to asequence set forth in SEQ ID NOS:28, 30, or 32. In certain embodiment,the HisRS polypeptide of the invention is not a polypeptide consistingof residues 1-48 of the full length human HisRS protein. These and otherHisRS polypeptides are included within the AARS polypeptides of thepresent invention.

Tryptophanyl-tRNA synthetases (WRS), also referred to as tryptophan-tRNAligases, belong to the class I tRNA synthetase family. Tryptophanyl-tRNAsynthetase catalyzes the aminoacylation of tRNA^(trp) with tryptophan,an essential function in protein synthesis. Human WRS has a kinasedomain in the N-terminal region and a serine phosphorylation site nearthe C-terminus.

Two main forms of human tryptophanyl-tRNA synthetase are produced invivo through alternative mRNA splicing, to yield the full-length protein(SEQ ID NO: 33), and a fragment thereof, often designated mini-WRS (SEQID NO:107). Also included are human T1-WRS (SEQ ID NO:108) and T2-WRS(SEQ ID NO:34), alternate splice variants that are produced from anIFN-gamma-sensitive promoter, the latter being an N-terminally truncatedfragment of WRS, as well as an N-terminal fragment (F1; SEQ ID NO:106)and fragment of WRS referred to as “Tolstrup” (SEQ ID NO:35). Othersplice variants of human WRS are known in the art (see, e.g., Liu etal., Nucleic Acids Research, 32(2):719-27, 2004, herein incorporated byreference).

Structurally, full-length WRS contains three parts, a canonicaldinucleotide-binding fold, a dimer interface, and a helical domain. Thisenzyme has enough structural homology to tyrosyl-tRNA synthetase (YRS)that the two enzymes can be described as conformational isomers.Structural elements interacting with the activated amino acid,tryptophanyl-5′ AMP, are almost exactly as seen in the tyrosyl-5′ AMPcomplex. Also, side chains that recognize indole are also highlyconserved, and require reorientation of a “specificity-determining”helix containing a conserved aspartate to assure selection of tryptophanversus tyrosine. The carboxy terminus, which is disordered and thereforenot seen in YRS, forms part of the dimer interface in WRS (see Doublieet al., Structure. 3:17-31, 1995).

The crystal structure of human T2-WRS has been reported at 2.5 Aresolution. This variant shares a very low sequence homology of 22% withBacillus stearothermophilus WRS (bWRS), however their overall structuresare strikingly similar. Structural comparison of T2-WRS with bWRSreveals substantial structural differences in the substrate-bindingpocket and at the entrance to the pocket that play important roles insubstrate binding and tRNA binding. T2-WRS has a wide opening to theactive site and adopts a compact conformation similar to the closedconformation of bWRS. Modeling studies indicate that tRNA binds with thedimeric enzyme and interacts primarily with the connective polypeptide 1of human WRS via its acceptor arm and the α-helical domain of WRS viaits anticodon loop.

The amino acid sequence of the full-length WRS polypeptide (or the mainsplice variant) is shown in SEQ ID NO:33. The amino acid sequence ofvarious splice variants or fragments are shown in SEQ ID NOS:34 and 35.Accordingly, these and other variants or fragments of WRS polypeptidesare included within the AARS polypeptides of the present invention.

Glutaminyl-tRNA synthetases (QRS) belong to the class I tRNA synthetasefamily, and the human protein is one of several mammalian aminoacyl-tRNAsynthetases that form a macromolecular protein complex. Theeukaryote-specific N-terminal appendix of QRS appears to stabilize theassociation of other components in the multi-ARS complex, whereas theC-terminal catalytic domain is necessary for QRS association with themulti-AARS complex.

The human QRS enzyme differs from both the bacterial and yeast enzymes,suggesting that a considerable part of human QRS has evolved to performfunctions other than the charging of tRNA. For instance, at least twodistinct regions (part I and part II) within the eukaryotic QRS (EC6.1.1.18)N-terminal region have no counterpart in Escherichia coli. Eventhough these regions are thought to bind RNA in a non-specific manner,enhancing interactions between the tRNA and enzyme, they are notessential for enzyme function (see, e.g., Wang et al., J. Biol. Chem.274:16508-12, 1999). Further, human and mouse cells express at least oneQRS variant that contains a deletion in part 1 of the N-terminal region,possibly due to an alternate start codon or alternate splicing. However,the available sequence data for yeast suggests that these microorganismsdo not express such a QRS variant, but rather only express a QRSpolypeptide that contains both part I and part II of the N-terminalregion.

Molecular phylogenetic studies of QRS suggest that it has relativelyrecently evolved from the closely related enzyme glutamyl-tRNAsynthetase. As evidence, selected glutaminyl-tRNA synthetase mutantsdisplay enhanced glutamic acid recognition. For instance, mutagenesis oftwo residues proximal to the active site, Phe-90 and Tyr-240, improvesglutamic acid recognition 3-5-fold in vitro and results in themisacylation of tRNA^(gln) with glutamic acid.

QRS has been crystallised in a variety of complexes, most importantlywith its cognate tRNA^(gln). The enzyme makes extensive contacts withthe concave face of the tRNA, and makes specific interactions with theCUG anticodon at positions 34 to 36, and with the base pairs between the5′ end and the 3′ end of the tRNA, just before the aminoacyl acceptor.

Certain QRS polypeptides possess anti-apoptotic activities. Forinstance, human QRS interacts with Fas ligation activated apoptosissignal-regulating kinase 1 (ASK1) in a glutamine-dependent manner. Thisinteraction involves the catalytic domains of the two enzymes, and isdissociated by Fas ligand. This interaction also inhibits both ASK1activity, as measured by in vitro kinase and transcription assays, andcell death induced by ASK1, an effect that is weakened by glutaminedeprivation. The anti-apoptotic interaction of QRS with ASK1 istherefore enhanced by the cellular concentration of glutamine andreduced by Fas ligation. This anti-apoptotic activity is believed to liein the C-terminal 539 amino acids of human QRS.

The amino acid sequence of the full-length QRS polypeptide is shown inSEQ ID NO:25. Certain specific examples of QRS variants, truncations, orfragments include QRS polypeptides that comprise or consist essentiallyof amino acids 1-183 (QRS1 or Q1), 1-220 (QRS2 or Q2), 1-249 (QRS3 orQ3), 1-200 (QRS4 or Q4), 1-(181-293), e.g., 1-180, 1-181, 1-182, 1-183,1-184, 1-185, 1-186, 1-187, 1-188, 1-189, 1-190, 1-191, 1-192, 1-193,1-194, 1-195, 1-196, 1-197, 1-198, 1-199, 1-200, etc., of SEQ ID NO:25(see Table 2). Also included are peptides of SEQ ID NOS:36-103 and109-115. Accordingly, these and other variants of QRS polypeptides areincluded within the AARS polypeptides of the present invention.

Glycyl-tRNA synthetase (GlyRS) is an α2 dimer that belongs to the classII family of tRNA synthetases (see, e.g., U.S. application Ser. No.12/492,925, herein incorporated by reference). The approximately 2462 bycDNA for this gene contains a large open reading frame (ORF) encoding685 amino acids with predicted M(r)=77,507 Da. The protein sequence ofhuman GlyRS has approximately 60% identity with B. mori GlyRS and 45%identity with S. cerevisiae GlyRS, and contains motifs 2 and 3characteristic of Class II tRNA synthetases

The amino acid sequence of the full-length GlyRS polypeptide is shown inSEQ ID NO:16. SEQ ID NOS:18-24 represent illustrative peptide sequencesanalyzed in determining GlyRS fragment boundaries.

Certain examples of GlyRS proteolytic fragments include polypeptidesthat comprise, consist essentially of, or consist of amino acid residues57-685, 214-685, 239-685, 311-685, 439-685, 511-658, 214-438, 367-438,214-420, 214-338, 85-127 1-213, 1-61, 85-214, 333-685, 128-685, 265-685,483-685 or 25-56 of SEQ ID NO:16, including biologically activetruncations or variants thereof (e.g., variants having about 80%, 85%,90%, 95%, 98% sequence identity to the fragments) that substantiallyretain at least one non-canonical biological activity of interest. Incertain specific embodiments, the GlyRS polypeptide is not a polypeptideas set forth in any one of NCBI # CR594947, U09587 and/or U09510.Accordingly, these and other variants of GlyRS polypeptides are includedwithin the AARS polypeptides of the present invention.

Additional examples of AARS polypeptides having non-canonical activitiesinclude phenylalanyl-tRNA synthetase (PheRS) splice variant polypeptides(PheRS_SV1P) (SEQ ID NO:104), which have a unique amino acid sequence inthe C-terminal end that is different from the full-length human PheRSprotein sequence, including variants and fragments of those PheRSpolypeptides; and aspartyl-tRNA synthetase (AspRS) polypeptides (SEQ IDNO:105), including fragments thereof that consist essentially of aminoacid residues 1-154, 1-174, 1-31, 399-425, 413-476 or 397-425 of SEQ IDNO:105.

Embodiments of the present invention contemplate the use of compositionscomprising AARS polypeptides, including truncated fragments, splicevariants, proteolytic fragments, and variants and/or modifiedpolypeptides thereof, for modulating inflammation in a subject. Includedare AARS polypeptides that reduce migration of immune cells such asgranulocytes to the lung, desensitize immune cells such as granulocytesto a given antigen or irritant, or both, among otherinflammatory-modulating activities described herein and known in theart. Variant proteins encompassed by the present application arebiologically active, that is, they continue to possess the inflammatoryresponse-modulating activity of a reference AARS polypeptide sequence(e.g., SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, 14, 16, 25, 28, 30, and32-108, 109-115 etc.). Such variants may result from, for example,genetic polymorphism or from human manipulation.

Biologically active variants of a reference AARS polypeptide fragmentwill have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%,usually about 90% to 95% or more, and typically about 98% or moresequence similarity or identity with the amino acid sequence of areference protein as determined by sequence alignment programs describedelsewhere herein using default parameters. A biologically active variantof a reference AARS polypeptide may differ from that protein generallyby as much 200, 100, 50 or 20 amino acid residues or suitably by as fewas 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5,as few as 4, 3, 2, or even 1 amino acid residue. In some embodiments, anAARS polypeptide differs from the reference sequences in SEQ ID NOS: 1,2, 3, 6, 8, 10, 12, 14, 16, 25, 28, 30, 32-108, or 109-115 by at leastone but by less than 15, 10 or 5 amino acid residues. In otherembodiments, it differs from the reference sequences in SEQ ID NOS: 1,2, 3, 6, 8, 10, 12, 14, 16, 25, 28, 30, 32-108, or 109-115 by at leastone residue but less than 20%, 15%, 10% or 5% of the residues.

An AARS polypeptide may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of a truncated and/or variant AARS polypeptide can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example, Kunkel(1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987,Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J.D. et al., (“Molecular Biology of the Gene”, Fourth Edition,Benjamin/Cummings, Menlo Park, Calif., 1987) and the references citedtherein. Guidance as to appropriate amino acid substitutions that do notaffect biological activity of the protein of interest may be found inthe model of Dayhoff et al., (1978) Atlas of Protein Sequence andStructure (Natl. Biomed. Res. Found., Washington, D.C.). Methods forscreening gene products of combinatorial libraries made by pointmutations or truncation, and for screening cDNA libraries for geneproducts having a selected property are known in the art. Such methodsare adaptable for rapid screening of the gene libraries generated bycombinatorial mutagenesis of AARS polypeptides. Recursive ensemblemutagenesis (REM), a technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify AARS polypeptide variants (Arkin andYourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave etal., (1993) Protein Engineering, 6: 327-331). Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be desirable as discussed in more detail below.

Biologically active truncated and/or variant AARS polypeptides maycontain conservative amino acid substitutions at various locations alongtheir sequence, as compared to a reference AARS amino acid sequence(e.g., SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, 14, 16, 25, 28, 30, 32-108,and 109-115). A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art, which can be generallysub-classified as follows:

Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH Amino acids having an acidic side chain include glutamic acid andaspartic acid.

Basic: The residue has a positive charge due to association with H ionat physiological pH or within one or two pH units thereof (e.g.,histidine) and the residue is attracted by aqueous solution so as toseek the surface positions in the conformation of a peptide in which itis contained when the peptide is in aqueous medium at physiological pH.Amino acids having a basic side chain include arginine, lysine andhistidine.

Charged: The residues are charged at physiological pH and, therefore,include amino acids having acidic or basic side chains (i.e., glutamicacid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and theresidue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium. Amino acids having a hydrophobic sidechain include tyrosine, valine, isoleucine, leucine, methionine,phenylalanine and tryptophan.

Neutral/polar: The residues are not charged at physiological pH, but theresidue is not sufficiently repelled by aqueous solutions so that itwould seek inner positions in the conformation of a peptide in which itis contained when the peptide is in aqueous medium. Amino acids having aneutral/polar side chain include asparagine, glutamine, cysteine,histidine, serine and threonine.

This description also characterizes certain amino acids as “small” sincetheir side chains are not sufficiently large, even if polar groups arelacking, to confer hydrophobicity. With the exception of proline,“small” amino acids are those with four carbons or less when at leastone polar group is on the side chain and three carbons or less when not.Amino acids having a small side chain include glycine, serine, alanineand threonine. The gene-encoded secondary amino acid proline is aspecial case due to its known effects on the secondary conformation ofpeptide chains. The structure of proline differs from all the othernaturally-occurring amino acids in that its side chain is bonded to thenitrogen of the α-amino group, as well as the α-carbon. Several aminoacid similarity matrices are known in the art (see e.g., PAM120 matrixand PAM250 matrix as disclosed for example by Dayhoff et al., 1978, Amodel of evolutionary change in proteins). Matrices for determiningdistance relationships In M. O. Dayhoff, (ed.), Atlas of proteinsequence and structure, Vol. 5, pp. 345-358, National BiomedicalResearch Foundation, Washington D.C.; and by Gonnet et al., (Science,256: 14430-1445, 1992), however, include proline in the same group asglycine, serine, alanine and threonine. Accordingly, for the purposes ofthe present invention, proline is classified as a “small” amino acid.

The degree of attraction or repulsion required for classification aspolar or nonpolar is arbitrary and, therefore, amino acids specificallycontemplated by the invention have been classified as one or the other.Most amino acids not specifically named can be classified on the basisof known behaviour.

Amino acid residues can be further sub-classified as cyclic ornon-cyclic, and aromatic or non-aromatic, self-explanatoryclassifications with respect to the side-chain substituent groups of theresidues, and as small or large. The residue is considered small if itcontains a total of four carbon atoms or less, inclusive of the carboxylcarbon, provided an additional polar substituent is present; three orless if not. Small residues are, of course, always non-aromatic.Dependent on their structural properties, amino acid residues may fallin two or more classes. For the naturally-occurring protein amino acids,sub-classification according to this scheme is presented in Table A.

TABLE A Amino acid sub-classification Sub-classes Amino acids AcidicAspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine; Cyclic:Histidine Charged Aspartic acid, Glutamic acid, Arginine, Lysine,Histidine Small Glycine, Serine, Alanine, Threonine, ProlinePolar/neutral Asparagine, Histidine, Glutamine, Cysteine, Serine,Threonine Polar/large Asparagine, Glutamine Hydrophobic Tyrosine,Valine, Isoleucine, Leucine, Methionine, Phenylalanine, TryptophanAromatic Tryptophan, Tyrosine, Phenylalanine Residues that influenceGlycine and Proline chain orientation

Conservative amino acid substitution also includes groupings based onside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulphur-containing side chains is cysteineand methionine. For example, it is reasonable to expect that replacementof a leucine with an isoleucine or valine, an aspartate with aglutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid will not have a majoreffect on the properties of the resulting variant polypeptide. Whetheran amino acid change results in a functional truncated and/or variantAARS polypeptide can readily be determined by assaying its activity, asdescribed herein (see, e.g., Examples 1, 2, 10, and 11). Conservativesubstitutions are shown in Table B under the heading of exemplarysubstitutions. Amino acid substitutions falling within the scope of theinvention, are, in general, accomplished by selecting substitutions thatdo not differ significantly in their effect on maintaining (a) thestructure of the peptide backbone in the area of the substitution, (b)the charge or hydrophobicity of the molecule at the target site, (c) thebulk of the side chain, or (d) the biological function. After thesubstitutions are introduced, the variants are screened for biologicalactivity.

TABLE B Exemplary Amino Acid Substitutions Original Residue ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn,His, Lys, Asn Glu Asp, Lys Asp Gly Pro Pro His Asn, Gln, Lys, Arg ArgIle Leu, Val, Met, Ala, Phe, Norleu Leu Leu Norleu, Ile, Val, Met, Ala,Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Ile, Phe Leu Phe Leu, Val, Ile,Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe,Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleu Leu

Alternatively, similar amino acids for making conservative substitutionscan be grouped into three categories based on the identity of the sidechains. The first group includes glutamic acid, aspartic acid, arginine,lysine, histidine, which all have charged side chains; the second groupincludes glycine, serine, threonine, cysteine, tyrosine, glutamine,asparagine; and the third group includes leucine, isoleucine, valine,alanine, proline, phenylalanine, tryptophan, methionine, as described inZubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).

Thus, a predicted non-essential amino acid residue in a truncated and/orvariant AARS polypeptide is typically replaced with another amino acidresidue from the same side chain family. Alternatively, mutations can beintroduced randomly along all or part of an AARS coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor an activity of the parent polypeptide to identify mutants whichretain that activity. Following mutagenesis of the coding sequences, theencoded peptide can be expressed recombinantly and the activity of thepeptide can be determined A “non-essential” amino acid residue is aresidue that can be altered from the reference sequence of an embodimentpolypeptide without abolishing or substantially altering one or more ofits activities. Suitably, the alteration does not substantially abolishone of these activities, for example, the activity is at least 20%, 40%,60%, 70% or 80% 100%, 500%, 1000% or more of a reference AARSpolypeptide. An “essential” amino acid residue is a residue that, whenaltered from the reference AARS polypeptide, results in abolition of anactivity of the parent molecule such that less than 20% of the referenceactivity is present. For example, such essential amino acid residuesinclude those that are conserved in AARS polypeptides across differentspecies, including those sequences that are conserved in the activebinding site(s) or motif(s) of AARS polypeptides from various sources.

Accordingly, the present invention also contemplates variants of thenaturally-occurring AARS polypeptide sequences or theirbiologically-active fragments, wherein the variants are distinguishedfrom the naturally-occurring sequence by the addition, deletion, orsubstitution of one or more amino acid residues. In general, variantswill display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90,91, 92, 93, 94, 95, 96, 97, 98, 99% similarity or sequence identity to areference AARS polypeptide sequence, for example, as set forth in SEQ IDNOS: 1, 2, 3, 6, 8, 10, 12, 14, 16, 25, 28, 30, 32-108, and 109-115.Moreover, sequences differing from the native or parent sequences by theaddition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150 or more amino acids but which retain theproperties of a parent or reference AARS polypeptide sequence arecontemplated. In certain embodiments, the C-terminal or N-terminalregion of any AARS polypeptide, including the AARS polypeptides of SEQID NOS: 1, 2, 3, 6, 8, 10, 12, 14, 16, 25, 28, 30, 32-108, or 109-115,may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 ormore amino acids, or by about 10-50, 20-50, 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600,600-650, 650-700 or more amino acids, including all integers and rangesin between (e.g., 101, 102, 103, 104, 105), so long as the truncatedAARS polypeptide is capable of modulating an inflammatory response,either in vivo, in vitro, or ex vivo (e.g., reducing migration of immunecells such as granulocytes, including neutrophils and eosinophils).

In some embodiments, variant polypeptides differ from a reference AARSsequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6,5, 4, 3 or 2 amino acid residue(s). In other embodiments, variantpolypeptides differ from the corresponding sequences of SEQ ID NOS: 1,2, 3, 6, 8, 10, 12, 14, 16, 25, 28, 30, 32-108, or 109-115 by at least1% but less than 20%, 15%, 10% or 5% of the residues. (If thiscomparison requires alignment, the sequences should be aligned formaximum similarity. “Looped” out sequences from deletions or insertions,or mismatches, are considered differences.) The differences are,suitably, differences or changes at a non-essential residue or aconservative substitution.

In certain embodiments, a variant polypeptide includes an amino acidsequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more sequence identity orsimilarity to a corresponding sequence of an AARS polypeptide as, forexample, set forth in SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12, 14, 16, 25, 28,30, 32-108, or 109-115 and has the ability to reduce pulmonaryinflammation in a subject, such as by reducing the migration orrecruitment of neutrophils or eosinophils to the lung.

Calculations of sequence similarity or sequence identity betweensequences (the terms are used interchangeably herein) are performed asfollows. To determine the percent identity of two amino acid sequences,or of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In certain embodiments, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, 60%, and even more preferably atleast 70%, 80%, 90%, 100% of the length of the reference sequence. Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch,(1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blossum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller (1989,Cabios, 4: 11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997, Nucleic Acids Res, 25:3389-3402). When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused.

Variants of an AARS polypeptide can be identified by screeningcombinatorial libraries of mutants of an AARS polypeptide. Libraries orfragments e.g., N terminal, C terminal, or internal fragments, of AARSprotein coding sequence can be used to generate a variegated populationof fragments for screening and subsequent selection of variants of anAARS polypeptide.

Methods for screening gene products of combinatorial libraries made bypoint mutation or truncation, and for screening cDNA libraries for geneproducts having a selected property are known in the art. Such methodsare adaptable for rapid screening of the gene libraries generated bycombinatorial mutagenesis of AARS polypeptides.

Also included are proteolytic fragments of AARS polypeptides. In certainillustrative embodiments, proteolytic fragments of AARS polypeptides maybe produced using a variety of proteolytic enzymes or proteolyticchemical agents, according to techniques known and available in the art.Proteolytic fragments can be produced in vitro, such as by incubatingAARS polypeptides with one or more proteases (as described herein andknown in the art) under controlled conditions and isolating andcharacterizing the fragments produced therefrom. Proteolytic fragmentscan also be produced in vivo, or endogenously, such as by recombinantlyexpressing the AARS polypeptides in a selected cell (e.g., bacterialcell, eukaryotic cell), and isolating and characterizing the endogenousfragments produced therefrom (see, e.g., Example 10).

Proteases are usually classified according to three major criteria: (i)the reaction catalysed, (ii) the chemical nature of the catalytic site,and (iii) the evolutionary relationship, as revealed by the structure.General examples of proteases or proteinases, as classified by mechanismof catalysis, include aspartic proteases, serine proteases, cysteineproteases, and metalloproteases.

Most aspartic proteases belong to the pepsin family. This familyincludes digestive enzymes, such as pepsin and chymosin, as well aslysosomal cathepsins D and processing enzymes such as renin, and certainfungal proteases (e.g., penicillopepsin, rhizopuspepsin,endothiapepsin). A second family of aspartic proteases includes viralproteinases such as the protease from the AIDS virus (HIV), also calledretropepsin.

Serine proteases include two distinct families. First, the chymotrypsinfamily, which includes the mammalian enzymes such as chymotrypsin,trypsin, elastase, and kallikrein, and second, the substilisin family,which includes the bacterial enzymes such as subtilisin. The general 3Dstructure between these two families is different, but they have thesame active site geometry, and catalysis proceeds via the samemechanism. The serine proteases exhibit different substratespecificities, differences which relate mainly to amino acidsubstitutions in the various enzyme subsites (substrate residueinteracting sites). Some serine proteases have an extended interactionsite with the substrate whereas others have a specificity that isrestricted to the P1 substrate residue.

The cysteine protease family includes the plant proteases such aspapain, actinidin, and bromelain, several mammalian lysosomalcathepsins, the cytosolic calpains (calcium-activated), as well asseveral parasitic proteases (e.g., Trypanosoma, Schistosoma). Papain isthe archetype and the best studied member of the family. Recentelucidation of the X-ray structure of the Interleukin-1-beta ConvertingEnzyme has revealed a novel type of fold for cysteine proteinases.

The metalloproteases are one of the older classes of proteases, found inbacteria, fungi, and higher organisms. They differ widely in theirsequences and their 3D structures, but the great majority of enzymescontain a zinc atom that is catalytically active. In some cases, zincmay be replaced by another metal such as cobalt or nickel without lossof proteolytic activity. Bacterial thermolysin has been wellcharacterized and its crystallographic structure indicates that zinc isbound by two histidines and one glutamic acid. Many metalloproteasescontain the sequence motif HEXXH, which provides two histidine ligandsfor the zinc. The third ligand is either a glutamic acid (thermolysin,neprilysin, alanyl aminopeptidase) or a histidine (astacin, serralysin).

Illustrative proteases include, for example, achromopeptidase,aminopeptidase, ancrod, angiotensin converting enzyme, bromelain,calpain, calpain I, calpain II, carboxypeptidase A, carboxypeptidase B,carboxypeptidase G, carboxypeptidase P, carboxypeptidase W,carboxypeptidase Y, caspase 1, caspase 2, caspase 3, caspase 4, caspase5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11,caspase 12, caspase 13, cathepsin B, cathepsin C, cathepsin D, cathepsinE, cathepsin G, cathepsin H, cathepsin L, chymopapain, chymase,chymotrypsin, clostripain, collagenase, complement C1r, complement C1s,complement Factor D, complement factor I, cucumisin, dipeptidylpeptidase IV, elastase (leukocyte), elastase (pancreatic),endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C,endoproteinase Lys-C, enterokinase, factor Xa, ficin, furin, granzyme A,granzyme B, HIV Protease, IGase, kallikrein tissue, leucineaminopeptidase (general), leucine aminopeptidase (cytosol), leucineaminopeptidase (microsomal), matrix metalloprotease, methionineaminopeptidase, neutrase, papain, pepsin, plasmin, prolidase, pronase E,prostate specific antigen, protease alkalophilic from Streptomycesgriseus, protease from Aspergillus, protease from Aspergillus saitoi,protease from Aspergillus sojae, protease (B. licheniformis) (alkalineor alcalase), protease from Bacillus polymyxa, protease from Bacillussp, protease from Rhizopus sp., protease S, proteasomes, proteinase fromAspergillus oryzae, proteinase 3, proteinase A, proteinase K, protein C,pyroglutamate aminopeptidase, rennin, rennin, streptokinase, subtilisin,thermolysin, thrombin, tissue plasminogen activator, trypsin, tryptaseand urokinase.

Tables C-G illustrate the type of proteolytic fragments that can beproduced in vitro by incubating AARS polypeptides with variousproteases. In certain embodiments, the incubation conditions can becontrolled so that only certain cleavage sites are cleaved by theindicated protease, to achieve only partial cleavage, followed byisolation of the desired proteolytic fragment according to techniquesknown in the art (e.g., chromatography). Once a desired fragment hasbeen isolated and characterized (e.g., sequenced) according to routinetechniques in the art, it can be cloned and produced recombinantly, orproduced synthetically, as desired.

Hence, included within the AARS polypeptides of the invention are anyproteolytic fragments that can be produced by the exemplary proteases inTables C-G, in addition to the proteases listed elsewhere herein,including any combination of proteases (e.g., Caspase 1 andhydroxylamine), or any combination of individual cleavage sites. Also,the residue position of cleavage sites may be approximate. Merely by wayof illustration, an AARS proteolytic fragment may include about residues1-165, about residues 166-445, about residues 166-455, about residues166-716, about residues 445-716, or about residues 455-716 of GlyRS thathas been cleaved or partially cleaved by incubation with iodosobenzoicacid (see Table C). As an additional illustration, an AARS proteolyticfragment may include about residues 1-98, about residues 1-135, aboutresidues 98-135, about residues 1-234, about residues 98-234, aboutresidues 1-379, about residues 234-674, or about residues 135-737 of QRSthat has been cleaved or partially cleaved by proline-endopeptidase (seeTable D). As a further illustrative example, an AARS polypeptide mayinclude about residues 1-210, about residues 1-273, about residues1-295, about residues 210-273, about residues 210-295, about residues273-295 of QRS that has been cleaved or partially cleaved byhydroxylamine Similar patterns can be applied to any of the AARSpolypeptides and any of the proteases in Tables C-G, or to the otherproteases listed herein or known in the art.

TABLE C Glycyl-tRNA synthetase (EC 6.1.1.14) (Glycine-tRNA ligase)(GlyRS) Protease Position of cleavage sites (Residue No.) Arg-Cproteinase 5 10 13 23 27 33 34 52 68 72 79 101 103 121 130 131 166 213297 310 331 337 342 344 388 391 412 428 430 464 474 560 583 596 602 640656 657 660 687 689 693 696 722 Asp-N endopeptidase 55 75 83 89 91 115116 119 125 134 148 178 195 199 204 214 227 246 255 270 355 360 369 393424 442 445 462 467 510 522 553 598 647 648 661 672 674 687 689 707 717Asp-N endopeptidase + 55 60 61 75 82 83 89 91 96 105 108 115 116 119 124125 134 148 171 172 176 178 N-terminal Glu 185 195 199 204 209 214 227233 237 239 246 252 255 270 298 312 332 344 349 351 355 358 360 369 393396 424 433 435 442 445 447 456 462 467 482 487 497 510 516 522 523 528530 535 538 542 544 553 567 568 575 589 596 598 599 625 632 635 647 648661 662 672 674 687 689 697 700 707 717 719 726 729 734 737 738BNPS-Skatole 165 445 455 716 CNBr 1 55 124 182 202 226 239 281 292 348390 433 437 516 530 532 555 585 628 692 Caspase 1 215 Chymotrypsin-high132 133 134 138 141 148 150 165 169 198 201 212 249 258 261 278 282 285295 specificity (C termto 305 308 314 321 330 346 354 365 374 376 408409 414 416 429 440 445 453 455 [FYW], not before P) 467 497 508 518 526540 549 561 566 579 586 589 593 604 605 614 627 630 658 668 674 716 726Clostripain 5 10 13 23 27 3334 52 68 72 79 101 103 121 130 131 166 213297 310 331 337 342 344 388 391 412 428 430 464 474 560 583 596 602 640656 657 660 687 689 693 696 722 Formic acid 56 76 84 90 92 116 117 120126 135 149 179 196 200 205 215 228 247 256 271 356 361 370 394 425 443446 463 468 511 523 554 599 648 649 662 673 675 688 690 708 718 Glutamylendopeptidase 61 62 83 97 106 109 125 172 173 177 186 210 234 238 240253 299 313 333 345 350 352 359 397 434 436 448 457 483 488 498 517 524529 531 536 539 543 545 568 569 576 590 597 600 626 633 636 663 698 701720 727 730 735 738 739 Hydroxylamine 208 711 Iodosobenzoic acid 165 445455 716 LysC 80 82 85 93 99 102 108 115 123 129 158 190 197 204 207 219224 229 230 235 236 264 283 309 318 360 364 379 389 419 426 450 477 484487 490 501 506 509 510 513 537 547 553 559 563 615 632 646 679 733 734LysN 79 81 84 92 98 101 107 114 122 128 157 189 196 203 206 218 223 228229 234 235 263 282 308 317 359 363 378 388 418 425 449 476 483 486 489500 505 508 509 512 536 546 552 558 562 614 631 645 678 732 733 NTCB(2-nitro-5- 40 154 179 210 230 441 443 460 465 470 521 524 615thiocyanobenzoic acid) Proline-endopeptidase 6 28 298 363 485Staphylococcal peptidase 61 83 97 106 109 125 172 177 186 210 234 238240 253 299 313 333 345 350 352 I 359 397 434 436 448 457 483 488 498517 524 529 531 536 539 543 545 568 576 590 597 600 626 633 636 663 698701 720 727 730 735 738 Trypsin 10 13 23 33 34 52 68 72 79 80 82 85 9399 101 102 103 108 115 121 123 129 130 131 158 166 190 197 204 207 213219 224 229 230 235 236 264 283 309 310 318 331 337 342 344 360 364 379388 389 391 412 419 426 428 430 450 464 474 477 487 490 501 506 509 510513 537 547 553 559 560 563 583 596 602 615 632 640 646 656 657 660 679687 689 693 696 722 733 734

TABLE D Glutaminyl-tRNA synthetase (EC 6.1.1.18) (Glutamine-tRNA ligase)(QRS) Protease Positions of cleavage sites (Residue No.) Arg-Cproteinase 21 34 62 64 67 68 95 109 132 134 141 154 195 201 202 225 265267 301 351 352 361 376 378 391 403 419 427 463 464 486 497 509 515 523524 525 538 558 567 576 616 629 639 666 667 690 694 745 764 Asp-Nendopeptidase 4 48 64 99 102 105 160 169 183 199 205 214 302 303 319 336339 376 409 413 429 438 445 474 509 511 512 558 562 588 597 617 668 702713 723 728 738 751 753 770 Asp-N endopeptidase + 4 16 21 34 48 64 83 9199 102 105 107 109 119 122 123 126 139 151 160 167 169 N-terminal Glu181 183 185 196 197 199 205 208 211 214 221 226 235 257 270 302 303 307309 310 319 336 339 347 362 363 376 380 381 387 396 398 408 409 413 429438 445 448 458 474 482 509 511 512 529 548 553 558 562 572 588 597 598614 617 620 621 623 645 658 661 668 671 687 692 701 702 705 713 723 728738 743 751 753 769 770 BNPS-Skatole 159 324 345 375 432 469 482 511 632680 CNBr 1 146 150 164 171 221 250 321 380 390 404 408 413 548 569 686Caspase1 184 Chymotrypsin-high 10 57 71 75 93 107 142 144 159 189 231238 243 286 288 290 299 302 314 315 324 specificity (C termto 327 330334 338 339 343 345 356 375 387 395 418 422 432 438 440 460 467 468[FYW], not before P) 469 477 482 484 491 511 517 535 603 608 613 619 627632 643 677 680 692 696 711 738 741 743 748 749 762 Clostripain 21 34 6264 67 68 95 109 132 134 141 154 195 201 202 225 265 267 301 351 352 361376 378 391 403 419 427 463 464 486 497 509 515 523 524 525 538 558 567576 616 629 639 666 667 690 694 745 764 Formic acid 5 49 65 100 103 106161 170 184 200 206 215 303 304 320 337 340 377 410 414 430 439 446 475510 512 513 559 563 589 598 618 669 703 714 724 729 739 752 754 771Glutamyl endopeptidase 17 22 35 84 92 108 110 120 123 124 127 140 152168 182 186 197 198 209 212 222 227 236 258 271 308 310 311 348 363 364381 382 388 397 399 409 449 459 483 530 549 554 573 599 615 621 622 624646 659 662 672 688 693 702 706 744 770 Hydroxylamine 210 273 295Iodosobenzoic acid 159 324 345 375 432 469 482 511 632 680 LysC 19 25 5079 80 158 163 166 180 187 188 190 193 205 230 233 239 254 282 292 309313 331 366 392 394 405 412 421 431 458 496 498 586 601 620 628 652 673675 699 736 740 759 769 774 LysN 18 24 49 78 79 157 162 165 179 186 187189 192 204 229 232 238 253 281 291 308 312 330 365 391 393 404 411 420430 457 495 497 585 600 619 627 651 672 674 698 735 739 758 768 773 NTCB(2-nitro-5- 110 297 318 357 432 442 444 455 470 477 535 555 656 664 686729 thiocyanobenzoic acid) Proline-endopeptidase 98 135 234 379 674 737Staphylococcal peptidase 17 22 35 84 92 108 110 120 123 127 140 152 168182 186 197 209 212 222 227 236 I 258 271 308 310 348 363 381 388 397399 409 449 459 483 530 549 554 573 599 615 621 624 646 659 662 672 688693 702 706 744 770 Thrombin 567 Trypsin 19 21 25 34 50 62 64 67 68 7980 95 109 132 141 154 158 163 166 180 187 188 190 193 195 201 202 205225 230 239 254 265 267 282 292 301 309 313 331 351 352 361 366 376 391392 394 403 405 412 419 421 427 431 458 463 464 486 496 497 498 509 515523 525 538 558 567 576 586 601 616 620 628 629 639 652 666 667 675 690694 699 740 745 759 764 769 774

TABLE E Tryptophanyl-tRNA synthetase, cytoplasmic (EC 6.1.1.2)(Tryptophan-tRNA ligase) (WRS) (Interferon-induced protein 53) (IFP53)(hWRS) Protease Positions of cleavage sites (Residue No.) Arg-Cproteinase 24 106 119 122 127 133 134 141 162 298 300 318 321 326 381388 417 448 449 464 Asp-N endopeptidase 33 36 56 60 75 82 85 98 100 112141 147 184 196 197 204 208 220 227 236 238 270 272 298 301 311 313 321353 362 381 394 396 408 409 410 418 453 468 Asp-N endopeptidase + N- 410 20 33 34 3655 56 60 75 78 80 81 82 85 98 100 112 114 120 141 147 150terminal Glu 166 184 196 197 198 204 208 216 220 227 236 238 270 272 298301 311 313 321 353 362 381 384 385 394 396 407 408 409 410 413 418 428435 443 450 453 454 458 468 BNPS-Skatole 88 182 203 CNBr 1 42 48 143 169195 241 243 319 350 401 425 461 Caspase1 61 363 Chymotrypsin-high 13 5058 84 88 100 107 131 137 138 150 156 157 159 177 179 182 187 201 203specificity (C-term to 212 214 227 233 235 240 247 248 260 267 269 289297 316 317 339 360 377 [FYW], not before P) 390 400 402 405 406 420 460468 470 Clostripain 24 106 119 122 127 133 134 141 162 298 300 318 321326 381 388 417 448 449 464 Enterokinase 200 412 Formic acid 34 37 57 6176 83 86 99 101 113 142 148 185 197 198 205 209 221 228 237 239 271 273299 302 312 314 322 354 363 382 395 397 409 410 411 419 454 469 Glutamylendopeptidase 5 11 21 35 56 79 81 82 115 121 151 167 199 217 385 386 408414 429 436 444 451 455 459 Iodosobenzoic acid 88 182 203 LysC 27 33 4147 51 59 96 102 111 114 153 154 181 200 204 220 231 249 253 256 264 277331 349 366 369 371 374 412 418 431 432 450 458 465 LysN 26 32 40 46 5058 95 101 110 113 152 153 180 199 203 219 230 248 252 255 263 276 330348 365 368 370 373 411 417 430 431 449 457 464 NTCB (2-nitro-5- 61 224273 304 308 393 thiocyanobenzoic acid) Proline-endopeptidase 128 155 332Staphylococcal peptidase I 5 11 21 35 56 79 81 115 121 151 167 199 217385 408 414 429 436 444 451 455 459 Thrombin 162 326 Trypsin 24 27 33 4147 51 59 96 102 106 111 114 119 122 133 134 141 153 162 181 200 204 220231 249 253 256 264 277 298 300 318 321 326 349 366 369 371 374 381 388412 417 418 431 432 448 449 450 458 464 465

TABLE F Tyrosyl-tRNA synthetase (EC 6.1.1.1) (Tyrosyl-tRNA ligase) (YRS)Protease Positions of cleavage sites (Residue No.) Arg-C proteinase 1634 93 135 189 207 237 279 325 367 371 400 418 432 450 Asp-Nendopeptidase 2 60 74 80 121 131 143 172 179 186 232 235 239 279 293 297307 321 342 368 382 384 392 416 455 477 493 Asp-N endopeptidase + 2 7 819 23 24 28 32 34 60 67 74 80 87 90 97 105 112 121 127 131 143 150 156N-terminal Glu 172 173 174 179 186 195 226 227 228 232 235 238 239 250255 273 279 280 293 295 297 301 307 313 321 325 342 358 360 361 368 378382 384 389 392 395 397 412 413 416 434 445 452 455 464 472 477 478 479488 493 498 499 BNPS-Skatole 40 87 283 505 CNBr 1 56 83 104 211 214 223350 431 439 511 Caspase1 75 494 Chymotrypsin-high 39 40 52 53 62 73 7987 96 97 117 123 129 134 176 183 192 194 198 204 249 263 specificity (Ctermto 275 283 289 292 299 328 388 409 468 472 488 495 505 510 [FYW],not before P) Clostripain 16 34 93 135 189 207 237 279 325 367 371 400418 432 450 Formic acid 3 61 75 81 122 132 144 173 180 187 233 236 240280 294 298 308 322 343 369 383 385 393 417 456 478 494 Glutamyl 8 9 2024 25 29 33 35 68 88 91 98 106 113 128 151 157 174 175 196 227 228 229endopeptidase 239 251 256 274 281 296 302 314 326 359 361 362 379 390396 398 413 414 435 446 453 465 473 479 480 489 499 500 Hydroxylamine258 Iodosobenzoic acid 40 87 283 505 LysC 10 26 28 32 37 47 58 64 84 102114 116 119 127 146 147 154 178 190 197 206 222 231 238 242 243 244 246247 265 272 282 287 297 310 319 327 334 335 346 348 352 356 374 380 391412 427 430 470 474 482 484 485 486 490 496 506 513 520 523 LysN 9 25 2731 36 46 57 63 83 101 113 115 118 126 145 146 153 177 189 196 205 221230 237 241 242 243 245 246 264 271 281 286 296 309 318 326 333 334 345347 351 355 373 379 390 411 426 429 469 473 481 483 484 485 489 495 505512 519 522 NTCB (2-nitro-5- 66 249 423 441 500 518 thiocyanobenzoicacid) Proline-endopeptidase 48 159 306 349 382 428 483 Staphylococcal 820 24 29 33 35 68 88 91 98 106 113 128 151 157 174 196 227 239 251 256274 peptidase I 281 296 302 314 326 359 361 379 390 396 398 413 435 446453 465 473 479 489 499 Trypsin 10 16 26 28 32 34 37 58 64 84 93 102 114116 119 127 135 146 147 154 178 189 190 197 206 207 222 231 237 238 242243 244 246 247 265 272 279 282 287 297 310 319 325 327 334 335 346 352356 367 371 374 380 391 400 412 418 430 432 450 470 474 484 485 486 490496 506 513 520 523

TABLE G Histidyl-tRNA synthetase (EC 6.1.1.21) (Histidine-tRNA ligase)(HisRS) Protease Positions of cleavage sites (Residue No.) Arg-Cproteinase 4 17 19 63 68 73 82 86 128 137 149 157 158 165 167 169 214215 232 266 326 362 375 388 396 405 424 479 484 490 491 500 501 Asp-Nendopeptidase 47 63 77 92 109 115 118 129 158 174 176 182 187 205 212217 227 238 241 264 268 285 300 314 315 320 328 363 370 432 472 487 492Asp-N endopeptidase + N- 2 7 8 15 28 31 32 33 47 48 63 73 77 89 92 97100 108 109 115 118 122 129 158 terminal Glu 169 174 176 182 187 189 196205 212 217 227 238 241 246 247 251 255 261 264 268 280 285 296 300 306314 315 320 328 336 348 349 363 370 386 393 397 400 401 407 421 422 429432 438 455 456 467 469 472 484 485 487 491 492 495 496 BNPS-Skatole 246432 CNBr 1 70 104 141 163 185 195 220 253 369 Chymotrypsin-high 54 65 7784 97 107 115 129 135 138 150 156 168 171 172 176 182 207 221 231specificity (C term to 246 270 306 308 312 320 330 331 336 363 370 390432 442 454 [FYW], not before P) Clostripain 4 17 19 63 68 73 82 86 128137 149 157 158 165 167 169 214 215 232 266 326 362 375 388 396 405 424479 484 490 491 500 501 Formic acid 48 64 78 93 110 116 119 130 159 175177 183 188 206 213 218 228 239 242 265 269 286 301 315 316 321 329 364371 433 473 488 493 Glutamyl endopeptidase 3 8 9 16 29 32 33 34 49 74 9098 101 109 123 170 190 197 247 248 252 256 262 281 297 307 337 349 350387 394 398 401 402 408 422 423 430 439 456 457 468 470 485 486 492 496497 Iodosobenzoic acid 246 432 LysC 12 22 25 37 40 42 51 53 57 60 75 85100 106 112 118 143 148 154 193 210 230 240 243 250 257 288 293 303 317373 376 403 418 419 426 437 443 444 447 472 477 499 LysN 11 21 24 36 3941 50 52 56 59 74 84 99 105 111 117 142 147 153 192 209 229 239 242 249256 287 292 302 316 372 375 402 417 418 425 436 442 443 446 471 476 498NTCB (2-nitro-5- 82 173 190 195 223 234 378 454 506 508 thiocyanobenzoicacid) Staphylococcal peptidase I 3 8 16 29 32 49 74 90 98 101 109 123170 190 197 247 252 256 262 281 297 307 337 349 387 394 398 401 408 422430 439 456 468 470 485 492 496 Trypsin 4 12 17 19 22 25 37 40 42 51 5357 60 63 68 73 75 82 85 86 100 106 112 118 128 137 143 148 149 154 157158 165 167 169 193 210 214 215 230 232 240 243 250 257 266 288 293 303317 326 362 373 375 376 388 396 403 405 418 419 424 426 437 443 444 447472 477 479 484 490 491 499 500 501

Certain embodiments relate to isolated AARS polypeptides, comprising,consisting essentially of, or consisting of amino acid sequences thathave been derived from endogenous, naturally-occurring AARS polypeptidefragments, and pharmaceutical compositions comprising said fragments,and methods of use thereof. In certain embodiments, as noted above, thesequences of naturally-occurring endogenous proteolytic fragments can begenerated or identified, for instance, from various cellular fractions(e.g., cytosolic, membrane, nuclear) and/or conditioned medium fromvarious cell-types, including primary cells and cell lines. Examples ofsuch cell types include, without limitation, immune cells such asmonocytes, dendritic cells, macrophages (e.g., RAW 264.7 macrophages;see Example 5), neutrophils, eosinophils, basophils, and lymphocytes,such as B-cells and T-cells (e.g., CD4+ helper and CD8+ killer cells),including primary T-cells and T-cell lines such as Jurkat T-cells, aswell as natural killer (NK) cells.

In certain embodiments, endogenous proteolytic fragments can beidentified by techniques such as mass-spectrometry, or equivalenttechniques. Merely by way of illustration and not limitation, in certainembodiments the proteomes from various cell types or fractions thereofmay be separated by 1D SDS-PAGE and the gel lanes cut into bands atfixed intervals; after which the bands may be optionally digested withan appropriate protease, such as trypsin, to release the peptides, whichmay then be analyzed by 1D reverse phase LC-MS/MS. The resultingproteomic data may be integrated into so-called peptographs, which plot,in the left panel, sequence coverage for a given protein in thehorizontal dimension (N to C terminus, left to right) versus SDS-PAGEmigration in the vertical dimension (high to low molecular weight, topto bottom). The specific peptide fragments can then be sequenced ormapped. Table H provides a set of illustrative mouse QRS polypeptidefragments that were identified from RAW macrophages according to theseexemplary techniques. Table I provides the corresponding set of humanQRS polypeptide fragments. Table J provides a set of illustrative humanQRS polypeptide fragments that were identified from human JurkatT-cells.

TABLE H Mouse QRS Polypeptide Fragments SEQ ID PEPTIDE SEQUENCE NO:ETLKNEALSTQLR 36 EAATQAHQILGSTIDKATGVLLYDLVSR 37ETLKNEALSTQLREAATQAHQILGSTIDKATGVLLYDLVSR 38 DFEQECGVGVVVTPEQIEEAVESTINK39 FNMGLLMGEAR* 40 MIKNEVDMQVLHLLGPK* 41 NEVDMQVLHLLGPK* 42TPGYVITPYTMDLLK 43 FDDTNPEKEEAK* 44 VEELKGHNPLPSPWR 45 DRPKEESLLLFEAMR46 VEELKGHNPLPSPWRDRPKEESLLLFEAMR 47 LVMEDGKMDPVAYR* 48 VYCPVQWEYGR* 49ILQLVAAGAVR 50 DVLNDAAPRAMAVLEPLQVVITNFPAPK 51GFHQVPFASTVFIERSDFKEESEPGYKRLASGQPVGLR 52 AFIHWVSQPLVCEIR 53LGYFSVDPDSHQGQIVFNR 54 TPGYVITPYTMDLLK 55 AINFNFGYAK* 56 FDDTNPEKEEAK*57 FFTAIYDMVTWLGYTPYK 58 FDDTNPEKEEAKFFTAIYDMVTWLGYTPYK 59DRPKEESLLLFEAMR 60 VYCPVQWEYGR* 61 LNLHYAVVSK* 62 VYCPVQWEYGRLNLHYAVVSK*63 ILQLVAAGAVR 64 AMAVLEPLQVVITNFPAPK 65 PLDIRVPNFPADETK 66AMAVLEPLQVVITNFPAPKPLDIRVPNFPADETK 67SDFKEESEPGYKRLASGQPVGLRHTGYVIELQNIVR 68 AFIHWVSQPLVCEIR 69LGYFSVDPDSHQGQIVFNR 70 KATGVLLYDLVSR 71 SFLVSYIANK 72DFEQECGVGVVVTPEQIEEAVESTINK 73 MIKNEVDMQVLHLLGPK* 74EAATQAHQILGSTIDKATGVLLYDLVSR 75 *The mouse and human sequences areidentical.

TABLE I Human QRS Polypeptide Fragments SEQ ID PEPTIDE SEQUENCE NO:ETLKNSALSAQLR  76 EAATQAQQTLGSTIDKATGILLYGLASR  77ETLKNSALSAQLREAATQAQQTLGSTIDKATGILLYGLASR  78DFERECGVGVIVTPEQIEEAVEAAINR  79 TPGYVVTPHTMNLLK  80 GEELKGHNTLPSPWR  81DRPMEESLLLFEAMR  82 GEELKGHNTLPSPWRDRPMEESLLLFEAMR  83 ILQLVATGAVR  84DVLNDTAPRAMAVLESLRVIITNFPAAK  85 GFHQVPFAPIVFIERTDFKEEPEPGFKRLAWGQPVGLR 86 AFIHWVSQPLMCEVR  87 LGYFSVDPDSHQGKLVFNR  88 TPGYVVTPHTMNLLK  89FFTAICDMVAWLGYTPYK  90 FDDTNPEKEEAKFFTAIYDMVTWLGYTPYK  91DRPMEESLLLFEAMR  92 ILQLVATGAVR  93 AMAVLESLRVIITNFPAAK  94SLDIQVPNFPADETK  95 AMAVLESLRVIITNFPAAKSLDIQVPNFPADETK  96TDFKEEPEPGFKRLAWGQPVGLRHTGYVIELQHVVK  97 AFIHWVSQPLMCEVR  98LGYFSVDPDSHQGKLVFNR  99 KATGILLYGLASR 100 SFLVSYIASK 101DFERECGVGVIVTPEQIEEAVEAAINR 102 EAATQAQQTLGSTIDKATGILLYGLASR 103

TABLE J Human QRS Polypeptide Fragments from Jurkat T-cells SEQ IDPEPTIDE SEQUENCE NO: NSALSAQLREAATQAQQTLGSTIDK 109SHPLDPIDTVDFERECGVGVIVTPEQIEEAVEAAINR 110 LSFLVSYIASK 111ECGVGVIVTPEQIEEAVEAAINR 112 EAATQAQQTLGSTIDKATGILLYGLASR 113IHTEPQLSAALEYVR 114 NEVDMQVLHLLGPK 115

Hence, certain specific embodiments include isolated QRS polypeptidesthat comprise, consist essentially of, or consist of any one or more ofSEQ ID NOS:36-103 or 109-115 (in Tables H, I, and J above), whichmodulate inflammation, such as by reducing pulmonary inflammation,including variants thereof. In certain embodiments, these isolated QRSpolypeptide fragments may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the C-terminaland/or N-terminal residues that surround them, as characterized by theirlocation within the full-length QRS polypeptide. In certain embodiments,these isolated QRS polypeptide fragments may be truncated to contain 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20fewer of their C-terminal and/or N-terminal residues. Also included arepharmaceutical compositions comprising such QRS polypeptide fragments,and methods of using said polypeptides or compositions to treat asubject in need thereof.

The present invention also contemplates the use of AARS chimeric orfusion proteins for modulating inflammation. As used herein, an AARS“chimeric protein” or “fusion protein” includes an AARS polypeptide orpolypeptide fragment linked to either another AARS-polypeptide (e.g., tocreate multiple fragments), to a non-AARS polypeptide, or to both. A“non-AARS polypeptide” refers to a “heterologous polypeptide” having anamino acid sequence corresponding to a protein which is different froman AARS protein, and which is derived from the same or a differentorganism. The AARS polypeptide of the fusion protein can correspond toall or a portion of a biologically active AARS amino acid sequence. Incertain embodiments, an AARS fusion protein includes at least one (ortwo) biologically active portion of an AARS protein. The polypeptidesforming the fusion protein are typically linked C-terminus toN-terminus, although they can also be linked C-terminus to C-terminus,N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptidesof the fusion protein can be in any order.

The fusion partner may be designed and included for essentially anydesired purpose provided they do not adversely affect theinflammation-modulating activity of the polypeptide. For example, in oneembodiment, a fusion partner may comprise a sequence that assists inexpressing the protein (an expression enhancer) at higher yields thanthe native recombinant protein. Other fusion partners may be selected soas to increase the solubility of the protein or to enable the protein tobe targeted to desired intracellular compartments.

The fusion protein can include a moiety which has a high affinity for aligand. For example, the fusion protein can be a GST-AARS fusion proteinin which the AARS sequences are fused to the C-terminus of the GSTsequences. As another example, an AARS polypeptide may be fused to aneight amino acid tag at the C-terminus, such as an L-E-H-H-H-H-H-H (SEQID NO:5) tag. In certain specific embodiments, amino acids 1-364 of aYRS polypeptide are fused to a 365-L-E-H-H-H-H-H-H-372 (SEQ ID NO:5) tagat the C-terminus Such fusion proteins can facilitate the purificationand/or identification of an AARS polypeptide. Alternatively, the fusionprotein can be an AARS protein containing a heterologous signal sequenceat its N-terminus. In certain host cells, expression and/or secretion ofAARS proteins can be increased through use of a heterologous signalsequence.

More generally, fusion to heterologous sequences, such as an Fcfragment, may be utilized to remove unwanted characteristics or toimprove the desired characteristics (e.g., pharmacokinetic properties)of an AARS polypeptide. For example, fusion to a heterologous sequencemay increase chemical stability, decrease immunogenicity, improve invivo targeting, and/or increase half-life in circulation of an AARSpolypeptide.

Fusion to heterologous sequences may also be used to createbi-functional fusion proteins, such as bi-functional proteins that arenot only capable of reducing pulmonary inflammation through the AARSpolypeptide, but are also capable of modifying (i.e., stimulating orinhibiting) other pathways through the heterologous polypeptide.Examples of such pathways include, but are not limited to, variousimmune system-related pathways, such as innate or adaptive immuneactivation pathways, or cell-growth regulatory pathways, such asangiogenesis, or hematopoiesis. In certain aspects, the heterologouspolypeptide may act synergistically with the AARS polypeptide tomodulate inflammation-related pathways in a subject. Examples ofheterologous polypeptides that may be utilized to create a bi-functionalfusion protein include, but are not limited to, thrombopoietin,cytokines (e.g., IL-11), chemokines, and various hematopoietic growthfactors, in addition to biologically active fragments and/or variantsthereof.

Fusion proteins may generally be prepared using standard techniques. Forexample, DNA sequences encoding the polypeptide components of a desiredfusion may be assembled separately, and ligated into an appropriateexpression vector. The 3′ end of the DNA sequence encoding onepolypeptide component is ligated, with or without a peptide linker, tothe 5′ end of a DNA sequence encoding the second polypeptide componentso that the reading frames of the sequences are in phase. This permitstranslation into a single fusion protein that retains the biologicalactivity of both component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures, ifdesired. Such a peptide linker sequence is incorporated into the fusionprotein using standard techniques well known in the art. Certain peptidelinker sequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA83:8258 8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No.4,751,180. The linker sequence may generally be from 1 to about 50 aminoacids in length. Linker sequences are not required when the first andsecond polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences may be operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are typically located 5′ tothe DNA sequence encoding the first polypeptide. Similarly, stop codonsrequired to end translation and transcription termination signals arepresent 3′ to the DNA sequence encoding the second polypeptide.

In general, polypeptides and fusion polypeptides (as well as theirencoding polynucleotides) are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

Certain embodiments also encompass dimers of AARS polypeptides. Dimersmay include, for example, homodimers between two identical AARSpolypeptides, heterodimers between two different AARS polypeptides(e.g., a full-length YRS polypeptide and a truncated YRS polypeptide; atruncated YRS polypeptide and a truncated WRS polypeptide), and/orheterodimers between an AARS polypeptide and a heterologous polypeptide.Certain heterodimers, such as those between an AARS polypeptide and aheterologous polypeptide, may be bi-functional, as described herein.Also included are monomers of AARS polypeptides, including isolated AARSpolypeptides monomers that do not substantially dimerize with a secondAARS polypeptide, whether due to one or more substitutions, truncations,deletions, additions, chemical modifications, or a combination of thesealterations. In certain embodiments, monomeric AARS polypeptides possessbiological activities, including inflammatory response-modulatingactivities, which are not possessed by dimeric or multimeric AARSpolypeptide complexes.

Certain embodiments of the present invention also contemplate the use ofmodified AARS polypeptides, including modifications that improved thedesired characteristics of an AARS polypeptide, as described herein.Modifications of AARS polypeptides of the invention include chemicaland/or enzymatic derivatizations at one or more constituent amino acid,including side chain modifications, backbone modifications, and N- andC-terminal modifications including acetylation, hydroxylation,methylation, amidation, and the attachment of carbohydrate or lipidmoieties, cofactors, and the like. Exemplary modifications also includepegylation of an AARS-polypeptide (see, e.g., Veronese and Harris,Advanced Drug Delivery Reviews 54: 453-456, 2002, herein incorporated byreference).

In certain aspects, chemoselective ligation technology may be utilizedto modify truncated AARS polypeptides of the invention, such as byattaching polymers in a site-specific and controlled manner. Suchtechnology typically relies on the incorporation of chemoselectiveanchors into the protein backbone by either chemical or recombinantmeans, and subsequent modification with a polymer carrying acomplementary linker. As a result, the assembly process and the covalentstructure of the resulting protein—polymer conjugate may be controlled,enabling the rational optimization of drug properties, such as efficacyand pharmacokinetic properties (see, e.g., Kochendoerfer, CurrentOpinion in Chemical Biology 9:555-560, 2005).

The truncated and/or variant AARS polypeptides of the invention may beprepared by any suitable procedure known to those of skill in the art,such as by recombinant techniques. For example, AARS polypeptides may beprepared by a procedure including the steps of: (a) preparing aconstruct comprising a polynucleotide sequence that encodes a truncatedAARS polypeptide and that is operably linked to a regulatory element;(b) introducing the construct into a host cell; (c) culturing the hostcell to express the truncated AARS polypeptide; and (d) isolating thetruncated and/or variant AARS polypeptide from the host cell. Inillustrative examples, the nucleotide sequence encodes at least abiologically active portion of a polypeptide sequence set forth in, orderived from, SEQ ID NOS:1, 2, 3, 6, 8, 10, 12, or 14, or a biologicallyactive variant or fragment thereof. Recombinant AARS polypeptides can beconveniently prepared using standard protocols as described for examplein Sambrook, et al., (1989, supra), in particular Sections 16 and 17;Ausubel et al., (1994, supra), in particular Chapters 10 and 16; andColigan et al., Current Protocols in Protein Science (John Wiley & Sons,Inc. 1995-1997), in particular Chapters 1, 5 and 6.

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc.85:2149-2154 (1963)). Protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the desiredmolecule.

Polynucleotide Compositions

The present invention also provides isolated polynucleotides that encodethe aminoacyl-tRNA synthetase polypeptides of the invention, includingtruncations and/or variants thereof, as well as compositions comprisingsuch polynucleotides.

As used herein, the terms “DNA” and “polynucleotide” and “nucleic acid”refer to a DNA molecule that has been isolated free of total genomic DNAof a particular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

As will be understood by those skilled in the art, the polynucleotidesequences of this invention can include genomic sequences, extra-genomicand plasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an aminoacyl-tRNA synthetase or a portion thereof)or may comprise a variant, or a biological functional equivalent of sucha sequence. Polynucleotide variants may contain one or moresubstitutions, additions, deletions and/or insertions, as furtherdescribed below, preferably such that the inflammatoryresponse-modulating activity of the encoded polypeptide is notsubstantially diminished relative to the unmodified polypeptide. Theeffect on the inflammatory response-modulating activity of the encodedpolypeptide may generally be assessed as described herein.

In additional embodiments, the present invention provides isolatedpolynucleotides comprising various lengths of contiguous stretches ofsequence identical to or complementary to an aminoacyl-tRNA synthetase,wherein the isolated polynucleotides encode a truncated aminoacyl tRNAsynthetase as described herein.

Exemplary nucleotide sequences that encode the AARS polypeptides of theapplication encompass coding sequences, such as the polynucleotidesequences of SEQ ID NOS:4, 7, 9, 11, 13, 15, 17, 19, and 31, as well asportions of the full-length or substantially full-length nucleotidesequences of the AARS genes or their transcripts or DNA copies of thesetranscripts.

Portions of an AARS nucleotide sequence may encode polypeptide portionsor segments that retain the biological activity of the referencepolypeptide, including the polypeptides of SEQ ID NOS:1, 2, 3, 6, 8, 10,12, 14, 16, 25, 28, 30, 32-108, and 109-115 or polypeptides having anamino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, or 98%identical to these sequences. A portion of an AARS nucleotide sequencethat encodes a biologically active fragment of an AARS polypeptide mayencode at least about 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80,90, 100, 120, 150, 300 or 400 contiguous amino acid residues, or almostup to the total number of amino acids present in a full-length AARSpolypeptide. It will be readily understood that “intermediate lengths,”in this context and in all other contexts used herein, means any lengthbetween the quoted values, such as 101, 102, 103, etc.; 151, 152, 153,etc.; 201, 202, 203, etc.

The polynucleotides of the present invention, regardless of the lengthof the coding sequence itself, may be combined with other DNA sequences,such as promoters, polyadenylation signals, additional restrictionenzyme sites, multiple cloning sites, other coding segments, and thelike, such that their overall length may vary considerably. It istherefore contemplated that a polynucleotide fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol.

The invention also contemplates variants of the AARS nucleotidesequences. Nucleic acid variants can be naturally-occurring, such asallelic variants (same locus), homologs (different locus), and orthologs(different organism) or can be non naturally-occurring. Naturallyoccurring variants such as these can be identified with the use ofwell-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as known inthe art. Non-naturally occurring variants can be made by mutagenesistechniques, including those applied to polynucleotides, cells, ororganisms. The variants can contain nucleotide substitutions, deletions,inversions and insertions. Variation can occur in either or both thecoding and non-coding regions. The variations can produce bothconservative and non-conservative amino acid substitutions (as comparedin the encoded product). For nucleotide sequences, conservative variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the amino acid sequence of a reference AARS polypeptide,such as the sequences set forth in SEQ ID NOS: 1, 2, 3, 6, 8, 10, 12,14, 16, 25, 28, 30, 32-108, or 109-115. Variant nucleotide sequencesalso include synthetically derived nucleotide sequences, such as thosegenerated, for example, by using site-directed mutagenesis but whichstill encode an AARS polypeptide. Generally, variants of a particularAARS nucleotide sequence will have at least about 30%, 40% 50%, 55%,60%, 65%, 70%, generally at least about 75%, 80%, 85%, desirably about90% to 95% or more, and more suitably about 98% or more sequenceidentity to that particular nucleotide sequence as determined bysequence alignment programs described elsewhere herein using defaultparameters.

AARS nucleotide sequences can be used to isolate corresponding sequencesand alleles from other organisms, particularly other organisms ormicroorganisms. Methods are readily available in the art for thehybridization of nucleic acid sequences. Coding sequences from otherorganisms may be isolated according to well known techniques based ontheir sequence identity with the coding sequences set forth herein. Inthese techniques all or part of the known coding sequence is used as aprobe which selectively hybridizes to other AARS-coding sequencespresent in a population of cloned genomic DNA fragments or cDNAfragments (i.e., genomic or cDNA libraries) from a chosen organism.

Accordingly, the present invention also contemplates polynucleotidesthat hybridize to reference AARS nucleotide sequences, or to theircomplements, under stringency conditions described below. As usedherein, the term “hybridizes under low stringency, medium stringency,high stringency, or very high stringency conditions” describesconditions for hybridization and washing. Guidance for performinghybridization reactions can be found in Ausubel et al., (1998, supra),Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described inthat reference and either can be used. Reference herein to lowstringency conditions include and encompass from at least about 1% v/vto at least about 15% v/v formamide and from at least about 1 M to atleast about 2 M salt for hybridization at 42° C., and at least about 1 Mto at least about 2 M salt for washing at 42° C. Low stringencyconditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA,0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and (i)2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5%SDS for washing at room temperature. One embodiment of low stringencyconditions includes hybridization in 6×sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions). Medium stringency conditions includeand encompass from at least about 16% v/v to at least about 30% v/vformamide and from at least about 0.5 M to at least about 0.9 M salt forhybridization at 42° C., and at least about 0.1 M to at least about 0.2M salt for washing at 55° C. Medium stringency conditions also mayinclude 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2),7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii)0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at 60-65°C. One embodiment of medium stringency conditions includes hybridizingin 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC,0.1% SDS at 60° C. High stringency conditions include and encompass fromat least about 31% v/v to at least about 50% v/v formamide and fromabout 0.01 M to about 0.15 M salt for hybridization at 42° C., and about0.01 M to about 0.02 M salt for washing at 55° C. High stringencyconditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7%SDS for hybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS; or (ii) 0.5%BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at atemperature in excess of 65° C. One embodiment of high stringencyconditions includes hybridizing in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 65° C.

In certain embodiments, an AARS polypeptide is encoded by apolynucleotide that hybridizes to a disclosed nucleotide sequence undervery high stringency conditions. One embodiment of very high stringencyconditions includes hybridizing in 0.5 M sodium phosphate, 7% SDS at 65°C., followed by one or more washes in 0.2×SSC, 1% SDS at 65° C.

Other stringency conditions are well known in the art and a skilledartisan will recognize that various factors can be manipulated tooptimize the specificity of the hybridization. Optimization of thestringency of the final washes can serve to ensure a high degree ofhybridization. For detailed examples, see Ausubel et al., supra at pages2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to1.104.

While stringent washes are typically carried out at temperatures fromabout 42° C. to 68° C., one skilled in the art will appreciate thatother temperatures may be suitable for stringent conditions. Maximumhybridization rate typically occurs at about 20° C. to 25° C. below theT_(m) for formation of a DNA-DNA hybrid. It is well known in the artthat the T_(m) is the melting temperature, or temperature at which twocomplementary polynucleotide sequences dissociate. Methods forestimating T_(m) are well known in the art (see Ausubel et al., supra atpage 2.10.8).

In general, the T_(m), of a perfectly matched duplex of DNA may bepredicted as an approximation by the formula: T_(m)=81.5+16.6 (log₁₀M)+0.41 (% G+C)−0.63 (% formamide)−(600/length) wherein: M is theconcentration of Na⁺, preferably in the range of 0.01 molar to 0.4molar; % G+C is the sum of guanosine and cytosine bases as a percentageof the total number of bases, within the range between 30% and 75% G+C;% formamide is the percent formamide concentration by volume; length isthe number of base pairs in the DNA duplex. The T_(m) of a duplex DNAdecreases by approximately 1° C. with every increase of 1% in the numberof randomly mismatched base pairs. Washing is generally carried out atT_(m)−15° C. for high stringency, or T_(m)−30° C. for moderatestringency.

In one example of a hybridization procedure, a membrane (e.g., anitrocellulose membrane or a nylon membrane) containing immobilized DNAis hybridized overnight at 42° C. in a hybridization buffer (50%deionized formamide, 5×SSC, 5×Denhardt's solution (0.1% ficoll, 0.1%polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200mg/mL denatured salmon sperm DNA) containing a labeled probe. Themembrane is then subjected to two sequential medium stringency washes(i.e., 2×SSC, 0.1% SDS for 15 min at 45° C., followed by 2×SSC, 0.1% SDSfor 15 min at 50° C.), followed by two sequential higher stringencywashes (i.e., 0.2×SSC, 0.1% SDS for 12 min at 55° C. followed by 0.2×SSCand 0.1% SDS solution for 12 min at 65-68° C.

Polynucleotides and fusions thereof may be prepared, manipulated and/orexpressed using any of a variety of well established techniques knownand available in the art. For example, polynucleotide sequences whichencode polypeptides of the invention, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of a truncated and/or variant aminoacyl-tRNA synthetasepolypeptide in appropriate host cells. Due to the inherent degeneracy ofthe genetic code, other DNA sequences that encode substantially the sameor a functionally equivalent amino acid sequence may be produced andthese sequences may be used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing,expression and/or activity of the gene product.

In order to express a desired polypeptide, a nucleotide sequenceencoding the polypeptide, or a functional equivalent, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,Current Protocols in Molecular Biology (1989).

A variety of expression vector/host systems are known and may beutilized to contain and express polynucleotide sequences. These include,but are not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thepBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, vectors which direct highlevel expression of fusion proteins that are readily purified may beused. Such vectors include, but are not limited to, the multifunctionalE. coli cloning and expression vectors such as BLUESCRIPT (Stratagene),in which the sequence encoding the polypeptide of interest may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem.264:5503 5509 (1989)); and the like. pGEX Vectors (Promega, Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al., Methods Enzymol. 153:516-544 (1987).

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680(1984); Broglie et al., Science 224:838-843 (1984); and Winter et al.,Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw Hill,Yearbook of Science and Technology, pp. 191-196 (1992)).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhardet al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer or immediate/earlycytomegalovirus (CMV) enhancer/promoter region, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf. et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) geneswhich can be employed in tk- or aprt-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70(1980)); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); andals or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. U.S.A. 85:8047-51 (1988)). The use of visible markers hasgained popularity with such markers as anthocyanins, β-glucuronidase andits substrate GUS, and luciferase and its substrate luciferin, beingwidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Methods Mol. Biol. 55:121-131(1995)).

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). These and otherassays are described, among other places, in Hampton et al., SerologicalMethods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med.158:1211-1216 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins.

Antibody Compositions, Fragments Thereof and Other Binding Agents

According to another aspect, the present invention further providesbinding agents, such as antibodies and antigen-binding fragmentsthereof, that exhibit immunological binding to a polypeptide disclosedherein, or to a portion, variant or derivative thereof, and methods ofusing same. Preferably, such binding agents are effective for modulatingone or more of the non-canonical activities mediated by an AARSpolypeptide of the invention, or for detecting the presence or absenceof selected AARS polypeptides (e.g., truncations, alternate splicevariants, mutants) in a sample, such as a biological sample obtainedfrom a subject.

For example, certain embodiments contemplate a method of identifying orcharacterizing an AARS polypeptide in a subject, comprising obtaining abiological sample from the subject, contacting the biological samplewith an antibody, or antigen-binding fragment thereof, wherein theantibody or antigen-binding fragment specifically binds to an AARSpolypeptide of the invention, and detecting the presence or absence ofthe bound antibody, or antigen-binding fragment thereof, therebyidentifying or characterizing the AARS polypeptide in the subject. Incertain aspects, the antibody, or antigen-binding fragment thereof,specifically binds to a certain variant or truncated AARS polypeptide,such as a selected AARS mutant or alternate splice variant, but does notspecifically bind to other AARS polypeptides, such as a full-length,wild type AARS polypeptide.

An antibody, or antigen-binding fragment thereof, is said to“specifically bind,” “immunologically bind,” and/or is “immunologicallyreactive” to a polypeptide of the invention if it reacts at a detectablelevel (within, for example, an ELISA assay) with the polypeptide, anddoes not react detectably with unrelated polypeptides under similarconditions.

Immunological binding, as used in this context, generally refers to thenon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (k_(on)) and the “off rateconstant” (k_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of k_(off)/k_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem.59:439-473.

An “antigen-binding site,” or “binding portion” of an antibody refers tothe part of the immunoglobulin molecule that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. Three highly divergent stretches within the V regions of theheavy and light chains are referred to as “hypervariable regions” whichare interposed between more conserved flanking stretches known as“framework regions,” or “FRs.” Thus, the term “FR” refers to amino acidsequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

A binding agent may be, for example, a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an polypeptide of interest may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, thesemethods involve the preparation of immortal cell lines capable ofproducing antibodies having the desired specificity (i.e., reactivitywith the polypeptide of interest). Such cell lines may be produced, forexample, from spleen cells obtained from an animal immunized asdescribed above. The spleen cells are then immortalized by, for example,fusion with a myeloma cell fusion partner, preferably one that issyngeneic with the immunized animal. A variety of fusion techniques maybe employed. For example, the spleen cells and myeloma cells may becombined with a nonionic detergent for a few minutes and then plated atlow density on a selective medium that supports the growth of hybridcells, but not myeloma cells. A preferred selection technique uses HAT(hypoxanthine, aminopterin, thymidine) selection. After a sufficienttime, usually about 1 to 2 weeks, colonies of hybrids are observed.Single colonies are selected and their culture supernatants tested forbinding activity against the polypeptide. Hybridomas having highreactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

A number of therapeutically useful molecules are known in the art whichcomprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)₂” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRs and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRs. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

As used herein, the terms “veneered FRs” and “recombinantly veneeredFRs” refer to the selective replacement of FR residues from, e.g., arodent heavy or light chain V region, with human FR residues in order toprovide a xenogeneic molecule comprising an antigen-binding site whichretains substantially all of the native FR polypeptide foldingstructure. Veneering techniques are based on the understanding that theligand binding characteristics of an antigen-binding site are determinedprimarily by the structure and relative disposition of the heavy andlight chain CDR sets within the antigen-binding surface. Davies et al.(1990) Ann. Rev. Biochem. 59:439-473. Thus, antigen binding specificitycan be preserved in a humanized antibody only wherein the CDRstructures, their interaction with each other, and their interactionwith the rest of the V region domains are carefully maintained. By usingveneering techniques, exterior (e.g., solvent-accessible) FR residueswhich are readily encountered by the immune system are selectivelyreplaced with human residues to provide a hybrid molecule that compriseseither a weakly immunogenic, or substantially non-immunogenic veneeredsurface.

In another embodiment of the invention, monoclonal antibodies of thepresent invention may be coupled to one or more agents of interest. Forexample, a therapeutic agent may be coupled (e.g., covalently bonded) toa suitable monoclonal antibody either directly or indirectly (e.g., viaa linker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used.

Modulation of Inflammatory Responses and Methods of Use

Embodiments of the present invention relate to the discovery thataminoacyl-tRNA synthetase (AARS) polypeptides, and variants thereof,modulate inflammation in a variety of useful ways, both in vitro and invivo. For instance, in certain embodiments, the AARS polypeptides of thepresent invention reduce an inflammatory response, such as by reducingthe migration or infiltration of immune cells into selected tissues,increasing the production of anti-inflammatory cytokines, or reducingthe production of pro-inflammatory cytokines, among other mechanisms. Incertain embodiments, the AARS polypeptides of the present inventionincrease or stimulate an inflammatory response, such as by increasingthe migration or infiltration of immune cells into selected tissues,increasing the production pro-inflammatory cytokines, or reducing theproduction of anti-inflammatory cytokines, among other mechanisms.

“Inflammation” refers generally to the biological response of tissues toharmful stimuli, such as pathogens, damaged cells (e.g., wounds), andirritants. The term “inflammatory response” refers to the specificmechanisms by which inflammation is achieved and regulated, including,merely by way of illustration, immune cell activation or migration,cytokine production, vasodilation, including kinin release,fibrinolysis, and coagulation, among others described herein and knownin the art. Ideally, inflammation is a protective attempt by the body toboth remove the injurious stimuli and initiate the healing process forthe affected tissue or tissues. In the absence of inflammation, woundsand infections would never heal, creating a situation in whichprogressive destruction of the tissue would threaten survival. On theother hand, excessive or chronic inflammation may associate with avariety of diseases, such as hay fever, atherosclerosis, and rheumatoidarthritis, among others described herein and known in the art.

AARS polypeptides of the invention may modulate acute inflammation,chronic inflammation, or both. Certain embodiments relate to increasingacute inflammation or acute inflammatory responses, and certainembodiments relate to increasing chronic inflammation or chronicinflammatory responses. Depending on the needs of the subject, certainembodiments relate to reducing acute inflammation or inflammatoryresponses, and certain embodiments relate to reducing chronicinflammation or chronic inflammatory responses.

Acute inflammation relates to the initial response of the body topresumably harmful stimuli and involves increased movement of plasma andleukocytes from the blood into the injured tissues. It is a short-termprocess, typically beginning within minutes or hours and ending upon theremoval of the injurious stimulus. Acute inflammation may becharacterized by any one or more of redness, increased heat, swelling,pain, and loss of function. Redness and heat are due mainly to increasedblood flow at body core temperature to the inflamed site, swelling iscaused by accumulation of fluid, pain is typically due to release ofchemicals that stimulate nerve endings, and loss of function hasmultiple causes.

Acute inflammatory responses are initiated mainly by local immune cells,such as resident macrophages, dendritic cells, histiocytes, Kuppfercells and mastocytes. At the onset of an infection, burn, or otherinjuries, these cells undergo activation and release inflammatorymediators responsible for the clinical signs of inflammation, such asvasoactive amines and eicosanoids. Vasodilation and its resultingincreased blood flow cause the redness and increased heat. Increasedpermeability of the blood vessels results in an exudation or leakage ofplasma proteins and fluid into the tissue, which creates swelling.Certain released mediators such as bradykinin increase sensitivity topain, and alter the blood vessels to permit the migration orextravasation of leukocytes, such as neutrophils, which typicallymigrate along a chemotactic gradient created by the local immune cells.

Acute inflammatory responses also includes one or more acellularbiochemical cascade systems, consisting of preformed plasma proteinsmodulate, which act in parallel to initiate and propagate theinflammatory response. These systems include the complement system,which is mainly activated by bacteria, and the coagulation andfibrinolysis systems, which are mainly activated by necrosis, such asthe type of tissue damage that is caused by certain infections, burns,or other trauma. Hence, AARS polypeptides may be used to modulate acuteinflammation, or any of one or more of the individual acute inflammatoryresponses.

Chronic inflammation, a prolonged and delayed inflammatory response, ischaracterized by a progressive shift in the type of cells that arepresent at the site of inflammation, and often leads to simultaneous ornear simultaneous destruction and healing of the tissue from theinflammatory process. At the cellular level, chronic inflammatoryresponses involve a variety of immune cells such as monocytes,macrophages, lymphocytes, plasma cells, and fibroblasts, though incontrast to acute inflammation, which is mediated mainly bygranulocytes, chronic inflammation is mainly mediated by mononuclearcells such as monocytes and lymphocytes. Chronic inflammation alsoinvolves a variety of inflammatory mediators, such as IFN-γ and othercytokines, growth factors, reactive oxygen species, and hydrolyticenzymes. Chronic inflammation may last for many months or years, and mayresult in undesired tissue destruction and fibrosis.

Clinical signs of chronic inflammation are dependent upon duration ofthe illness, inflammatory lesions, cause and anatomical area affected.(see, e.g., Kumar et al., Robbins Basic Pathology-8^(th) Ed., 2009Elsevier, London; Miller, L M, Pathology Lecture Notes, AtlanticVeterinary College, Charlottetown, PEI, Canada). Chronic inflammation isassociated with a variety of pathological conditions or diseases,including, for example, allergies, Alzheimer's disease, anemia, aorticvalve stenosis, arthritis such as rheumatoid arthritis andosteoarthritis, cancer, congestive heart failure, fibromyalgia,fibrosis, heart attack, kidney failure, lupus, pancreatitis, stroke,surgical complications, inflammatory lung disease, inflammatory boweldisease, atherosclerosis, and psoriasis, among others described hereinand known in the art. Hence, AARS polypeptides may be used to treat ormanage chronic inflammation, modulate any of one or more of theindividual chronic inflammatory responses, or treat any one or morediseases or conditions associated with chronic inflammation.

AARS polypeptides may also modulate proliferative inflammation, aninflammatory process characterized by an increase in the number oftissue cells. These can encompass skin conditions such as psoriasis,seborrhea or eczema, or can also be thought of in terms of cancers andabnormal growths especially in light of accumulating evidence based onmore efficient molecular methods to document even low grade chronicinflammation.

In certain embodiments, AARS polypeptides may modulate inflammatoryresponses at the cellular level, such as by modulating the activation,inflammatory molecule secretion (e.g., cytokine or kinin secretion),proliferation, activity, migration, or adhesion of various cellsinvolved in inflammation. Examples of such cells include immune cellsand vascular cells. Immune cells include, for example, granulocytes suchas neutrophils, eosinophils and basophils, macrophages/monocytes,lymphocytes such as B-cells, killer T-cells (i.e., CD8+ T-cells), helperT-cells (i.e., CD4+ T-cells, including T_(h)1 and T_(h)2 cells), naturalkiller cells, γδ T-cells, dendritic cells, and mast cells. Examples ofvascular cells include smooth muscle cells, endothelial cells, andfibroblasts. Also included are methods of modulating an inflammatorycondition associated with one or more immune cells or vascular cells,including neutrophil-mediated, macrophage-mediated, andlymphocyte-mediated inflammatory conditions.

In certain embodiments, AARS polypeptides may modulate the levels oractivity of inflammatory molecules, including plasma-derivedinflammatory molecules and cell-derived inflammatory molecules. Includedare pro-inflammatory molecules and anti-inflammatory molecules. Examplesof plasma-derived inflammatory molecules include, without limitation,proteins or molecules of any one or more of the complement system, kininsystem, coagulation system, and the fibrinolysis system. Examples ofmembers of the complement system include C1, which exists in blood serumas a molecular complex containing about 6 molecules of C1q, 2 moleculesof C1r, and 2 molecules of C1s, C2 (a and b), C3(a and B), C4 (a and b),C5, and the membrane attack complex of C5a, C5b, C6, C7, C8, and C9.Examples of the kinin system include bradykinin, kallidin, kallidreins,carboxypeptidases, angiotensin-converting enzyme, and neutralendopeptidase.

Examples of cell-derived inflammatory molecules include, withoutlimitation, enzymes contained within lysosome granules, vasoactiveamines, eicosanoids, cytokines, acute-phase proteins, and soluble gasessuch as nitric oxide. Vasoactive amines contain at least one aminogroup, and target blood vessels to alter their permeability or causevasodilation. Examples of vasoactive amines include histamine andserotonin. Eicosanoids refer to signaling molecules made by oxidation oftwenty-carbon essential fatty acids, and include prostaglandins,prostacyclins, thromboxanes, and leukotrienes.

Cytokines refer to a variety of substances that are secreted by immunecells, and include polypeptides and glycoproteins. Typically, cytokinesare categorized as either autocrine cytokines, which act on the sametype of cell from which the cytokine is secreted, or paracrinecytokines, which are restricted to acting on a different cell type fromwhich the cytokine is secreted. Examples of cytokines, examples of theirproducing cells, examples of their target cells, and exemplaryactivities are included in Tables J and K below.

TABLE K Cytokines Selected Immune Cytokines and Their ActivitiesCytokine Producing Cell Target Cell Activity GM-CSF Th cells progenitorcells growth and differentiation of monocytes and DC IL-1a monocytes Thcells co-stimulation IL-1b macrophages B cells maturation andproliferation B cells NK cells activation DC various inflammation, acutephase response, fever IL-2 Th1 cells activated T and B cells, growth,proliferation, NK cells activation IL-3 Th cells stem cells growth anddifferentiation NK cells mast cells growth and histamine release IL-4Th2 cells activated B cells proliferation and differentiation IgG₁ andIgE synthesis macrophages MHC Class II T cells proliferation IL-5 Th2cells activated B cells proliferation and differentiation IgA synthesisIL-6 monocytes activated B cells differentiation into plasma cellsmacrophages plasma cells antibody secretion Th2 cells stem cellsdifferentiation stromal cells various acute phase response IL-7 marrowstroma stem cells differentiation into progenitor B and thymus stroma Tcells IL-8 macrophages neutrophils chemotaxis endothelial cells IL-10Th2 cells macrophages cytokine production B cells activation IL-12macrophages activated Tc cells differentiation into CTL B cells (withIL-2) NK cells activation IFN-α leukocytes various viral replication MHCI expression IFN-β fibroblasts various viral replication MHC Iexpression IFN- Th1 cells, various Viral replication gamma Tc cells, NKcells macrophages MHC expression activated B cells Ig class switch toIgG_(2a) Th2 cells proliferation macrophages pathogen elimination MIP-1αmacrophages monocytes, T cells chemotaxis MIP-1β lymphocytes monocytes,T cells chemotaxis TGF-β T cells, monocytes monocytes, macrophageschemotaxis activated macrophages IL-1 synthesis activated B cells IgAsynthesis various proliferation TNF-α macrophages, mast cells,macrophages CAM and cytokine expression NK cells tumor cells cell deathTNF-β Th1 and Tc cells phagocytes phagocytosis, NO production tumorcells cell death

TABLE L Cytokines Old Name New Name ENA-78 CXCL5 GROα CXCL1 GROβ CXCL2GROγ CXCL3 PF4 CXCL4 IP-10 CXCL10 Mig CXCL9 I-TAC CXCL11 SDF-1α/β CXCL12BCA-1 CXCL13 CXCL16 BRAK CXCL14 MCP-1 CCL2 MCP-4 CCL13 MCP-3 CCL7 MCP-2CCL8 MIP-1β CCL4 MIP-1αS CCL3 MIP-1αP CCL3LI RANTES CCL5 MPIF-1 CCL23HCC-1 CCL14 HCC-2 CCL15 HCC-4 CCL16 Eotaxin CCL11 Eotaxin-2 CCL24Eotaxin-3 CCL26 TARC CCL17 MDC CCL22 MIP-3α CCL20 ELC CCL19 SLC CCL21I-309 CCL1 TECK CCL25 CTACK CCL27 MEC CCL28 PARC CCL18 Lymphotactin XCL1SCM-1β XCL2 Fractalkine CX3CL1

In certain embodiments, AARS polypeptides increase the levels of any oneor more of TNF-α, MIP-1b, IL-12(p40), KC, MIP-2, or IL-10. In certainembodiments, AARS polypeptides increase the secretion of at least one ofTNF-α and IL-10 by peripheral blood mononuclear cells (PBMCs), includingmonocytes, lymphocytes, or both. In certain embodiments, AARSpolypeptides increase the secretion of IL-2 by lymphocytes such asactivated T-cells. In certain embodiments, AARS polypeptides reduceTNF-α secretion by immune cells such as PBMCs, and in certainembodiments reduce lipopolysaccharide-induced TNF-α secretion by theseand other cells. In certain embodiments, AARS polypeptides reduce IL-12secretion by immune cells such as PBMCs, and in certain embodimentsreduce lipopolysaccharide induced IL-12 secretion by these and othercells.

Each cytokine typically has a corresponding cytokine receptor. Examplesof classes of cytokine receptors include, without limitation, receptorsfrom the immunoglobulin (Ig) superfamily, such as the IL-1 receptortypes, which share structural homology with immunoglobulins(antibodies), cell adhesion molecules, and even some cytokines, andreceptors from the hematopoietic growth factor family, such as the IL-2receptor family and the receptors for GM-CSF, IL-3, and IL-5, receptorsfrom the interferon (type 2) family, including receptors for IFN β andγ. Additional examples include receptors from the tumor necrosis factors(TNF) (type 3) family, which share a cysteine-rich common extracellularbinding domain and interact with several other non-cytokine ligands suchas CD40, CD27 and CD30, receptors from the seven transmembrane helixfamily, including G-protein coupled receptors, and chemokine receptorssuch as CXCR4 and CCR5, as well as receptors for IL-8, MIP-1 and RANTES.Hence, in certain embodiments, AARS polypeptides may modulate the levelsor activity of one or more selected cytokines, such as those in Tables Jand K, the levels or activity of one or more selected cytokinereceptors, the interaction between cytokines and their receptors, or anycombination thereof.

AARS polypeptides may also modulate levels or activity of acute-phaseproteins. Examples of acute-phase proteins include C-reactive protein,serum amyloid A, serum amyloid P, and vasopressin. In certain instances,expression of acute-phase proteins can cause a range of undesiredsystemic effects including amyloidosis, fever, increased blood pressure,decreased sweating, malaise, loss of appetite, and somnolence.Accordingly, AARS polypeptides may modulate the levels or activity ofacute-phase proteins, their systemic effects, or both.

In certain embodiments, AARS polypeptides modulate local inflammation,systemic inflammation, or both. In certain embodiments, AARS polypeptidemay reduce or maintain (i.e., prevent further increases) localinflammation or local inflammatory responses. In certain embodiments,depending on the needs of the subject, AARS polypeptides may increaselocal inflammation or local inflammatory responses. In certainembodiments, AARS polypeptides may reduce or maintain (i.e., preventfurther increases) systemic inflammation or systemic inflammatoryresponses. In certain embodiments, depending on the needs of thesubject, AARS polypeptides may increase systemic inflammation orsystemic inflammatory responses.

In certain embodiments, the modulation of inflammation or inflammatoryresponses can be associated with one or more tissues or organs.Non-limiting examples of such tissues or organs include skin (e.g.,dermis, epidermis, subcutaneous layer), hair follicles, nervous system(e.g., brain, spinal cord, peripheral nerves), auditory system orbalance organs (e.g., inner ear, middle ear, outer ear), respiratorysystem (e.g., nose, trachea, lungs), gastroesophogeal tissues, thegastrointestinal system (e.g., mouth, esophagus, stomach, smallintestines, large intestines, rectum), vascular system (e.g., heart,blood vessels and arteries), liver, gallbladder, lymphatic/immune system(e.g., lymph nodes, lymphoid follicles, spleen, thymus, bone marrow),uro-genital system (e.g., kidneys, ureter, bladder, urethra, cervix,Fallopian tubes, ovaries, uterus, vulva, prostate, bulbourethral glands,epidiymis, prostate, seminal vesicles, testicles), musculoskeletalsystem (e.g., skeletal muscles, smooth muscles, bone, cartilage,tendons, ligaments), adipose tissue, mammaries, and the endocrine system(e.g., hypothalamus, pituitary, thyroid, pancreas, adrenal glands).Accordingly, AARS polypeptides may be used to modulate inflammationassociated with any of these tissues or organs, such as to treatconditions or diseases that are associated with the inflammation ofthese tissues or organs.

As noted above, certain embodiments may employ AARS polypeptides toreduce or manage (i.e., prevent further increases) inflammation orinflammatory responses associated with particular tissues or organs.Included are inflammatory responses and conditions associated with theskin, including inflammation, infections, and cancers associated withthe dermal, epidermal, and subcutaneous layers of the skin. Examples ofskin-associated inflammatory conditions include, without limitation,dermatitis, such as psoriasis, irritant dermatitis, seborrheicdermatitis, atopic dermatitis (eczema), allergic contact dermatitis,thermal-induced dermatitis, drug-induced dermatitis, dyshidroticdermatitis, urticaria, autoimmune dermatitis, skin cancer such asmelanoma, and bullous dermatitis. Also included are bacterial, viral andparasitic infections, erythema multiforme, erythema nodosum, granulomaannulare, poison oak/poison ivy, and toxic epidermal necrolysis.

Certain embodiments relate to reducing inflammatory responses andconditions associated with the nervous system, including inflammation,infections, and cancer associated with the brain and spinal cord of thecentral nervous system, the peripheral nervous system, and the meninges.Expression of inflammatory mediators including complement, adhesionmolecules, cyclooxygenase enzymes and their products and cytokines isincreased in experimental and clinical neurodegenerative disease, andintervention studies in experimental animals suggest that several ofthese factors contribute directly to neuronal injury. For instance,specific cytokines, such as interleukin-1 (IL-1), have been implicatedheavily in acute neurodegeneration, such as stroke and head injury.

Examples of nervous system associated inflammatory conditions include,without limitation, meningitis (i.e., inflammation of the protectivemembranes covering the brain and spinal cord), myelitis,encaphaloymyelitis (e.g., myalgic encephalomyelitis, acute disseminatedencephalomyelitis, encephalomyelitis disseminata or multiple sclerosis,autoimmune encephalomyelitis), arachnoiditis (i.e., inflammation of thearachnoid, one of the membranes that surround and protect the nerves ofthe central nervous system), granuloma, drug-induced inflammation ormeningitis, neurodegenerative diseases such as Alzheimer's disease,stroke, HIV-dementia, encephalitis such viral encephalitis and bacterialencephalitis, parasitic infections, inflammatory demyeleniatingdisorders, and auto-immune disorders such as CD8+ T Cell-mediatedautoimmune diseases of the CNS. Additional examples include Parkinson'sdisease, myasthenia gravis, motor neuropathy, Guillain-Barre syndrome,autoimmune neuropathy, Lambert-Eaton myasthenic syndrome, paraneoplasticneurological disease, paraneoplastic cerebellar atrophy,non-paraneoplastic stiff man syndrome, progressive cerebellar atrophy,Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea,Gilles de la Tourette syndrome, autoimmune polyendocrinopathy, dysimmuneneuropathy, acquired neuromyotonia, arthrogryposis multiplex, opticneuritis, and stiff-man syndrome.

As noted above, also included is inflammation associated with infectionsof the nervous system. Specific examples of bacterial infectionsassociated with inflammation of the nervous system include, withoutlimitation, streptococcal infection such as group B streptococci (e.g.,subtypes III) and Streptococcus pneumoniae (e.g., serotypes 6, 9, 14, 18and 23), Escherichia coli (e.g., carrying K1 antigen), Listeriamonocytogenes (e.g., serotype IVb), neisserial infection such asNeisseria meningitidis (meningococcus), staphylococcal infection,heamophilus infection such as Haemophilus influenzae type B, Klebsiella,and Mycobacterium tuberculosis. Also included are infections bystaphylococci and pseudomonas and other Gram-negative bacilli, mainlywith respect to trauma to the skull, which gives bacteria in the nasalcavity the potential to enter the meningeal space, or in persons withcerebral shunt or related device (e.g., extraventricular drain, Ommayareservoir). Specific examples of viral infections associated withinflammation of the nervous system include, without limitation,enteroviruses, herpes simplex virus type 1 and 2, human T-lymphotrophicvirus, varicella zoster virus (chickenpox and shingles), mumps virus,human immunodeficiency virus (HIV), and lymphocytic choriomeningitisvirus (LCMV). Meningitis may also result from infection by spirochetessuch as Treponema pallidum (syphilis) and Borrelia burgdorferi (Lymedisease), parasites such as malaria (e.g., cerebral malaria), fungi suchas Cryptococcus neoformans, and ameoba such as Naegleria fowleri.

Meningitis or other forms of nervous system inflammation may alsoassociate with the spread of cancer to the meninges (malignantmeningitis), certain drugs such as non-steroidal anti-inflammatorydrugs, antibiotics and intravenous immunoglobulins, sarcoidosis (orneurosarcoidosis), connective tissue disorders such as systemic lupuserythematosus, and certain forms of vasculitis (inflammatory conditionsof the blood vessel wall) such as Behçet's disease. Epidermoid cysts anddermoid cysts may cause meningitis by releasing irritant matter into thesubarachnoid space. Accordingly, AARS polypeptides may be used to treator manage any one or more of these conditions.

Certain embodiments relate to reducing inflammatory responses andconditions associated with the auditory system or balance organs, suchas the inner ear, middle ear, and the outer ear. Examples of auditorysystem or balance organ associated inflammatory conditions include,without limitation, outer ear inflammation (e.g., ear infections),middle ear inflammation, which may lead to fluid build-up in thenormally air-filled space and associated conductive hearing loss,labyrinthitis, an inner ear infection or inflammation causing bothdizziness (vertigo) and hearing loss, vestibular neuronitis, aninfection of the vestibular nerve, generally viral, causing vertigo, andcochlear neuronitis, an infection of the cochlear nerve, generallyviral, causing sudden deafness but no vertigo. Recipients of cochlearimplants for hearing loss are at an increased risk of pneumococcalmeningitis and its associated inflammation.

Certain embodiments relate to reducing inflammatory responses andconditions associated with the respiratory system, includinginflammation, infections, and cancer associated with the nose, trachea,and lungs. Examples of respiratory system associated inflammatoryconditions include, without limitation, atopic asthma, non-atopicasthma, allergic asthma, atopic bronchial IgE-mediated asthma, bronchialasthma, essential asthma, true asthma, intrinsic asthma caused bypathophysiologic disturbances, extrinsic asthma caused by environmentalfactors, essential asthma of unknown or inapparent cause, non-atopicasthma, bronchitic asthma, emphysematous asthma, exercise-inducedasthma, allergen induced asthma, cold air induced asthma, occupationalasthma, infective asthma caused by bacterial, fungal, protozoal, orviral infection, non-allergic asthma, incipient asthma, wheezy infantsyndrome and bronchiolytis, chronic or acute bronchoconstriction,chronic bronchitis, small airways obstruction, and emphysema. Furtherexamples include obstructive or inflammatory airways diseases such aschronic eosinophilic pneumonia, chronic obstructive pulmonary disease(COPD), COPD that includes chronic bronchitis, pulmonary emphysema ordyspnea associated or not associated with COPD, COPD that ischaracterized by irreversible, progressive airways obstruction, andadult respiratory distress syndrome (ARDS).

Further examples of conditions associated with pulmonary inflammationinclude conditions related to exacerbation of airways hyper-reactivityconsequent to other drug therapy, airways disease that is associatedwith pulmonary hypertension, bronchitis such as acute bronchitis, acutelaryngotracheal bronchitis, arachidic bronchitis, catarrhal bronchitis,croupus bronchitis, dry bronchitis, infectious asthmatic bronchitis,productive bronchitis, staphylococcus or streptococcal bronchitis andvesicular bronchitis, acute lung injury, and bronchiectasis such ascylindric bronchiectasis, sacculated bronchiectasis, fusiformbronchiectasis, capillary bronchiectasis, cystic bronchiectasis, drybronchiectasis and follicular bronchiectasis.

COPD in particular refers to a group of lung diseases that block airflowand make it increasingly difficult for affected individuals to breathenormally. Emphysema and chronic bronchitis are the two main conditionswithin the group of COPD diseases, but COPD can also refer to damagecaused by chronic asthmatic bronchitis, among other conditions known inthe art. In most cases, damage to the airways eventually interferes withthe exchange of oxygen and carbon dioxide in the lungs. Standardtreatments focus mainly on controlling symptoms and minimizing furtherdamage.

Emphysema represents one aspect of COPD. Emphysema leads to inflammationwithin the fragile walls of the alveoli, which may destroy some of thewalls and elastic fibers, allowing small airways to collapse uponexhaling, and impairing airflow out of the lungs. Signs and symptoms ofemphysema include, for instance, shortness of breath, especially duringphysical activities, wheezing, and chest tightness.

Chronic bronchitis represents another aspect of COPD. Chronic bronchitisis characterized by an ongoing cough, and leads to inflammation andnarrowing of the bronchial tubes. This condition also causes increasedmucus production, which can further block the narrowed tubes. Chronicbronchitis occurs mainly in smokers, and is typically defined as a coughthat lasts for at least three months a year for two consecutive years.Signs and symptoms of chronic bronchitis include, for example, having toclear the throat first thing in the morning, especially for smokers, achronic cough that produces yellowish sputum, shortness of breath in thelater stages, and frequent respiratory infections.

As noted above, COPD refers primarily to obstruction in the lungsresulting from the two above-noted chronic lung conditions. However,many individuals with COPD have both of these conditions.

Chronic asthmatic bronchitis represents another aspect of COPD. Chronicasthmatic bronchitis is usually characterized as chronic bronchitiscombined with asthma (bronchospasm). Asthma may occur when inflamed andinfected secretions irritate the smooth muscles in the airways. Symptomsare similar to those of chronic bronchitis, but also includeintermittent, or even daily, episodes of wheezing.

In certain embodiments, COPD is ultimately caused by cigarette smoke andother irritants. In the vast majority of cases, the lung damage thatleads to COPD is caused by long-term cigarette smoking. However, otherirritants may cause COPD, including cigar smoke, secondhand smoke, pipesmoke, air pollution and certain occupational fumes. Gastroesophagealreflux disease (GERD), which occurs when stomach acids wash back up intothe esophagus, can not only aggravate COPD, but may even cause it insome individuals. In rare cases, COPD results from a genetic disorderthat causes low levels of a protein called alpha-1-antitrypsin. Hence,risk factors for COPD include exposure to tobacco smoke, occupationalexposure to dusts and chemicals (long-term exposure to chemical fumes,vapors and dusts irritates and inflames the lungs), gastroesophagealreflux disease (a severe form of acid reflux—the backflow of acid andother stomach contents into the esophagus), age (COPD develops slowlyover years, so most people are at least 40 years old when symptomsbegin), and genetics (a rare genetic disorder known asalpha-1-antitrypsin deficiency is the source of a few cases of COPD).

In certain embodiments, COPD may also have an autoimmune component. Forinstance, lung and peripheral blood T cells in patients with severeemphysema secrete Th1 cytokines and chemokines when stimulated withelastin peptides in vitro, and these patients have increasedanti-elastin antibody as compared to controls (see Goswami et al., TheJournal of Immunology. 178: 130.41, 2007). Also, IgG autoantibodies withavidity for pulmonary epithelium, and the potential to mediatecytotoxicity, are prevalent in patients with COPD (see Feghali-Bostwicket al., Am J Respir Crit Care Med. 177:156-63, 2008). Since autoreactiveimmune responses may be important in the etiology of this disease,including, for example, auto-reactive responses to self-antigens such aselastin, may play a role in COPD, the use of AARS polypeptides todesensitize immune cells to these antigens may reduce pulmonaryinflammation.

As noted above, certain embodiments relate to the use of AARSpolypeptides to desensitize immune cells to selected antigens, includingself antigens and foreign antigens, irritants, allergens, or infectiousagents related to pulmonary inflammation. By desensitizing these immunecells to a selected antigen, AARS polypeptides may reduce the migrationor recruitment of these cells to the lungs, and thereby reduceinflammation. Examples of immune cells include lymphocytes, monocytes,macrophages, dendritic cells, and granulocytes, such as neutrophils,eosinophils, and basophils. Examples of antigens include, withoutlimitation, smoke such as cigarette smoke, air pollution, fumes such asthe fumes from welding, dust, including silica dust and workplace dustsuch as those found in coal mining and gold mining, chemicals such ascadmium and isocyanates. Also included are known allergens andinfectious agents, such as bacterial and viral or antigens, includinglipopolysaccharide (LPS), which may exacerbate COPD in sensitiveindividuals.

In addition to others described herein, examples of self-antigensinclude, without limitation, receptor ligands, chemoattractants, andsignaling molecules. In certain embodiments, the response to the antigenor self-antigen signals via a CXCR-2 receptor. Without wishing to bebound by any one theory, certain AARS polypeptides may bind theirputative receptor on the surface of neutrophils, such as the CXCR2receptor, which then results in the desensitization of the receptor(i.e., the receptor is internalized and no longer be present at the cellsurface). In these and similar instances, there then exists a populationof circulating neutrophils that no longer respond to CXCR-2 ligands,such as IL-8. Since IL-8 is is produced as a result of cigarette smokein COPD, for example, the densitization of certain neutrophils to CXCR-2ligands such as IL-8 reduces their migration to the lung, and therebyreduces the inflammation associated with COPD, especially that caused bycigarette smoke.

Complications or associated symptoms of COPD may include increased riskof respiratory infections, high blood pressure, heart problems (e.g.,heart attacks, arrhythmias, cor pulmonale), lung cancer (smokers withchronic bronchitis are at a higher risk of developing lung cancer thanare smokers who don't have chronic bronchitis), pneumonia, pneumothorax,and depression, among others known in the art. Further examples includecough that produces mucus and may be streaked with blood, fatigue,frequent respiratory infections, headaches, shortness of breath(dyspnea) that worsens with mild activity, swelling of the ankles, feet,or legs, which affects both sides of the body, and wheezing. AARSpolypeptides may be used to reduce or manage the complications orsymptoms associated with COPD or other pulmonary conditions related toinflammation.

Subjects with COPD may be identified according to routine diagnostictechniques known in the art. For instance, pulmonary function tests,such as spirometry, measure how much air the lungs can hold and how fastan individual can blow the air out of their lungs. Spirometry can detectCOPD before the appearance of symptoms, and can also be used to trackdisease progression and monitor treatment. In addition, chest X-raysshow emphysema, one of the main causes of COPD, and may also rule outother lung problems or heart failure. In addition, arterial blood gasanalysis measures how effectively the lungs bring oxygen into the bloodand remove carbon dioxide, providing an indication of COPD. Sputumexamination, i.e., the analysis of the cells in the sputum, can identifythe cause of certain lung problems and help rule out certain lungcancers. Also, computerized tomography (CT) scan produceshighly-detailed images of the internal organs, which can help detectemphysema, and, thus, COPD.

As elsewhere herein, the amount of AARS polypeptide administered to asubject with COPD (or at risk for COPD) will depend on thecharacteristics of that subject, such as general health, age, sex, bodyweight, and tolerance to drugs, as well as the degree, severity, andtype of reaction to the polypeptide. For instance, in desensitizingimmune cells such as circulating neutrophils, multiple administrationsmay be utilized (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc), typically at adefined frequency (number of administrations per day, per week, permonth, etc).

Also included are combination therapies. For instance, one or more AARSpolypeptides can be utilized in combination with other treatments forpulmonary inflammation or COPD. Examples of such treatments included,without limitation, lifestyle changes, such as quitting or reducingsmoking or other exposure to lung irritants, lung rehabilitation, theuse of bronchodilators (e.g., ipratropium, tiotropium, salmeterol,formoterol), steroids such as corticosteroids, antibiotics, metered-doseinhalers (MDIs) and dry powder inhalers (DPIs), nebulizers, replacementgene therapy for alpha-1-antitrypsin deficiency, oxygen therapy, andsurgery, including bullectomy, lung volume reduction surgery, and lungtransplant.

Certain embodiments relate to reducing inflammatory responses andconditions associated the gastrointestinal system, includinginflammation, infections, and cancer associated with the mouth,esophagus, stomach, small intestines, large intestines, and rectum.“Gastrointestinal inflammation” as used herein refers to inflammation ofa mucosal layer of the gastrointestinal tract, and encompasses acute andchronic inflammatory conditions. Acute inflammation is generallycharacterized by a short time of onset and infiltration or influx ofneutrophils. Chronic inflammation is generally characterized by arelatively longer period of onset and infiltration or influx ofmononuclear cells. Chronic inflammation can also typically characterizedby periods of spontaneous remission and spontaneous occurrence. “Mucosallayer of the gastrointestinal tract” is meant to include mucosa of thebowel (including the small intestine and large intestine), rectum,stomach (gastric) lining, oral cavity, and the like.

“Chronic gastrointestinal inflammation” refers to inflammation of themucosal of the gastrointestinal tract that is characterized by arelatively longer period of onset, is long-lasting (e.g., from severaldays, weeks, months, or years and up to the life of the subject), and isoften associated with infiltration or influx of mononuclear cells, andcan be further associated with periods of spontaneous remission andspontaneous occurrence. “Chronic gastrointestinal inflammatoryconditions” (also referred to as “chronic gastrointestinal inflammatorydiseases”) having such chronic inflammation include, but are not limitedto, inflammatory bowel disease (IBD), colitis induced by environmentalinsults (e.g., gastrointestinal inflammation associated with atherapeutic regimen, such as chemotherapy, radiation therapy, and thelike), colitis in conditions such as chronic granulomatous disease (see,e.g., Schappi et al., Arch Dis Child. 84:147-151, 2001), celiac disease,celiac sprue (i.e., a heritable disease in which the intestinal liningis inflamed in response to the ingestion of a protein known as gluten),food allergies, gastritis, infectious gastritis or enterocolitis (e.g.,Helicobacter pylori-infected chronic active gastritis) and other formsof gastrointestinal inflammation caused by an infectious agent, andother like conditions.

As used herein, “inflammatory bowel disease” or “IBD” refers to any of avariety of diseases characterized by inflammation of all or part of theintestines. Examples of inflammatory bowel disease include, but are notlimited to, Crohn's disease and ulcerative colitis. The term IBDincludes pseudomembranous colitis, hemorrhagic colitis, hemolytic-uremicsyndrome colitis, collagenous colitis, ischemic colitis, radiationcolitis, drug and chemically induced colitis, diversion colitis,ulcerative colitis, irritable bowel syndrome, irritable colon syndromeand Crohn's disease; and within Crohn's disease all the subtypesincluding active, refractory, and fistulizing and Crohn's disease.Hence, AARS polypeptides may be employed to treat or manage any one ormore of these conditions.

Certain embodiments relate to reducing inflammatory responses andconditions associated with the vascular system, or vascularinflammation, such as inflammation associated with the blood vessels andthe heart. Examples of vascular system associated inflammatoryconditions include, without limitation, myocarditis, pericarditis,occlusive disease, atherosclerosis, myocardial infarction, thrombosis,Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome,anti-factor VIII autoimmune disease, necrotizing small vesselvasculitis, microscopic polyangiitis, Churg and Strauss syndrome,pauci-immune focal necrotizing glomerulonephritis, crescenticglomerulonephritis, antiphospholipid syndrome, antibody induced heartfailure, thrombocytopenic purpura, autoimmune hemolytic anemia, cardiacautoimmunity in Chagas' disease, and anti-helper T lymphocyteautoimmunity. Also included are endocarditis, or infection of the heartvalves with spread of small clusters of bacteria through thebloodstream, phlebitis or vasculitis, inflammation of one or more veins,and thrombophlebitis, vein inflammation related to a thrombus.Thrombophlebitis may occur repeatedly in different locations, and isthen referred to as thrombophlebitis migrans, or migratingthrombophlebitis. Phlebitis may associate with a variety of causes, suchas bacterial infection, exposure to chemical agents, such as irritatingor vesicant solutions, physical trauma from skin puncture such asmovement of a cannula into the vein during insertion, medications suchas Celebrex, Olanzepine, antidepressants, and others, and alcohol abuse.Certain embodiments may relate to treating or managing heartinflammation caused by any one or more of acute rheumatic fever,congenital toxoplasmosis, enterovirus antenatal infection, lyme disease,and rheumatic fever.

Certain embodiments relate to reducing inflammatory responses andconditions associated with the liver or gallbladder, including acute andchronic liver inflammation, and acute and chronic cholecystis. Examplesof liver or gallbladder associated inflammatory conditions include,without limitation, auto-immune hepatitis, viral hepatitis (e.g.,Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis Dvirus, Hepatitis E virus, mononucleosis, rubella, Epstein-Barr virus,and cytomegalovirus), other causes of hepatitis such as severe bacterialinfection, ameobic infections, medicines (e.g., agomelatine,allopurinol, amitryptyline, amiodarone, asathioprine, paracetamol,halothane, ibuprofen, indomethacin, isoniazid, rifampicin, pyrazinamide,ketoconazole, loratadine, methotrexate, methyldopa, minocycline,nifedipine, nitrofurantoin, phenytoin, valproic acid, troglitazone,zidovudine), toxins (e.g., alcohol, fungal toxins), and metabolicdisorders (e.g., Wilson's disease, a disorder of the body's coppermetabolism, haemochromatosis, disorder of the body's iron metabolism,non-alcoholic steatohepatitis, alpha 1-antitrypsin deficiency).Additional examples include non-alcoholic fatty liver disease, cirrhosissuch as primary biliary cirrhosis, obstructive jaundice, ischemichepatitis, and gall bladder disease.

Certain embodiments relate to reducing inflammatory responses andconditions associated with the lymphatic/immune system. Examples oflymphatic/immune system associated inflammatory conditions include,without limitation, auto-immune diseases, such as Chagas disease,chronic obstructive pulmonary disorder (COPD), Crohn's disease,dermatomyositis, diabetes mellitus type I, endometriosis, Goodpasture'ssyndrome, Graves' disease, Guillain-Barre syndrome, Hachimoto's disease,hidradenitis suppurativa, Kawasaki disease, IgA nephropathy, idiopathicthrombocytopenia purpura, interstitial cystitis, lupus erythematosus,mixed connective tissue disease, morphea, myasthenia gravis, narcolepsy,neuromyotonia, pemphigus vulgaris, pernicous anemia, psoriasis,psoriatic arthritis, poliomyositis, primary biliary cirrhosis,rheumatoid arthritis, schizophrenia, scleroderma, Sjogren's syndrome,stiff person syndrome, temporal arteritis, ulcerative colitis, vitiligo,and Wegener's granulomatosis, in addition to autoimmune hemolyticanemia, and various lymphadenopathies.

Also included are immune-related inflammatory conditions associated withthe transplantation of a graft, tissue, cell or organ, such as graftrejection, chronic graft rejection, subacute graft rejection, hyperacutegraft rejection, acute graft rejection, and graft versus host disease.In certain embodiments, AARS polypeptides can be administered to atransplant donor before or during tissue removal. In certainembodiments, AARS polypeptides can be administered to a transplantrecipient before, during, and/or after transplant therapy to reduceinflammation-related complications of transplant therapy. Examples oftransplant therapies include bone marrow, stem cell, peripheral blood,liver, lung, heart, skin, and kidney, among others known in the art.Additional examples include inflammatory conditions associated withallergies, such as asthma, hives, urticaria, pollen allergy, dust miteallergy, venom allergy, cosmetics allergy, latex allergy, chemicalallergy, drug allergy, insect bite allergy, animal dander allergy,stinging plant allergy, poison ivy allergy and food allergy.

Certain embodiments relate to reducing inflammatory responses andconditions associated with the uro-genital system. Examples ofuro-genital system associated inflammatory conditions include, withoutlimitation, inflammations, infections or cancers of the ureter, bladder,urethra, cervix, Fallopian tubes, ovaries, uterus, womb, vulva,prostate, bulbourethral glands, epidiymis, prostate, seminal vesicles,testicles, or kidneys. Also included are auto-immune interstitialnephritis, renal abscess (intrarenal or extrarenal), acute prostatitis,hematuria, urethritis (e.g., Chlamydia and other sexually transmitteddiseases), pelvic inflammatory disease (PID), and prostatic abscess.Also included is nephritis associated with one or more ofglomerulonephritis, lupus nephritis, nephropathy, gout, poisons orchemicals (e.g., ether, thallium sulfate), certain medications (e.g.,piroxicam, candyl, feldene gel, fensaid, pirox), Herrmann syndrome,yellow fever, immune complex diseases, typhoid fever, urethralstricture, renal tuberculosism, and post-streptococcalglomerulonephritis.

Certain embodiments relate to reducing inflammatory responses andconditions associated with the musculoskeletal system. Examples ofmusculoskeletal system associated inflammatory conditions include,without limitation, arthritis such as rheumatoid arthritis and psoriaticarthritis, ankylosing spondylitis, auto-immune myositis, primarySjogren's syndrome, smooth muscle auto-immune disease, myositis,polymyositis, tendinitis, ligament inflammation, cartilage inflammation,joint inflammation, synovial inflammation, carpal tunnel syndrome,chronic muscle inflammation, and bone inflammation, including boneinflammation associated with osteoporosis and osteoarthritis. Alsoincluded are Tietze's syndrome, a benign, painful, nonsuppurativelocalized swelling of the costosternal, sternoclavicular, orcostochondral joints, costochondritis, sternalis syndrome, xiphoidalgia,spontaneous sternoclavicular subluxation, sternocostoclavicularhyperostosis, fibromyalgia, shoulder tendinitis or bursitis, goutyarthritis, polymyalgia rheumatica, lupus erythematosus, bone spurs, andfractures such as stress fractures.

Certain embodiments relate to reducing inflammatory responses andconditions associated with the endocrine system. Examples of endocrinesystem associated inflammatory conditions include, without limitation,inflammation, infection, or cancer associated with the hypothalamus,pituitary, thyroid, pancreas, or adrenal glands, glandular diseases suchas pancreatic disease, diabetes such as Type I diabetes, thyroiddisease, Graves' disease, thyroiditis, spontaneous autoimmunethyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarianautoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitisand Type I autoimmune polyglandular syndrome.

Certain embodiments relate to reducing inflammatory responses andconditions associated with adipose tissues, an active participant inregulating physiologic and pathologic processes, including immunity andinflammation. Macrophages are components of adipose tissue and activelyparticipate in its activities. Furthermore, cross-talk betweenlymphocytes and adipocytes can lead to immune regulation. Adipose tissueproduces and releases a variety of pro-inflammatory andanti-inflammatory factors, including the adipokines leptin, adiponectin,resistin, and visfatin, as well as cytokines and chemokines, such asTNF-alpha, IL-6, monocyte chemoattractant protein 1, and others.Proinflammatory molecules produced by adipose tissue have beenimplicated as active participants in the development of insulinresistance and the increased risk of cardiovascular disease associatedwith obesity. In contrast, reduced leptin levels may predispose toincreased susceptibility to infection caused by reduced T-cell responsesin malnourished individuals. Altered adipokine levels have been observedin a variety of inflammatory conditions (see, e.g., Fantuzzi, J AllergyClin Immunol. 115:911-19, 2005; and Berg et al., Circulation Research.96:939, 2005).

AARS polypeptides may also be employed to treat or manage inflammationassociated with hypersensitivity. Examples of such conditions includetype I hypersensitivity, type II hypersensitivity, type IIIhypersensitivity, type IV hypersensitivity, immediate hypersensitivity,antibody mediated hypersensitivity, immune complex mediatedhypersensitivity, T-lymphocyte mediated hypersensitivity, and delayedtype hypersensitivity.

AARS polypeptides may also be employed to treat or manageauto-inflammatory conditions. Examples of auto-inflammatory conditionsinclude familial Mediterranean fever, TNF receptor associated periodicsyndrome (TRAPS), Hyper-IgD syndrome (HIDS), CIAS1-related diseases suchas Muckle-Wells syndrome, familial cold auto-inflammatory syndrome, andneonatal onset multisystem inflammatory disease, PAPA syndrome (pyogenicsterile arthritis, pyoderma gangrenosum, acne), and Blau syndrome.

AARS polypeptides may be employed to treat or manage inflammationassociated with a variety of cancers. Examples of such cancers include,without limitation, prostate cancer, breast cancer, colon cancer, rectalcancer, lung cancer, ovarian cancer, testicular cancer, stomach cancer,bladder cancer, pancreatic cancer, liver cancer, kidney cancer, braincancer, melanoma, non-melanoma skin cancer, bone cancer, lymphoma,leukemia, thyroid cancer, endometrial cancer, multiple myeloma, acutemyeloid leukemia, neuroblastoma, glioblastoma, and non-Hodgkin'slymphoma.

As noted above, certain embodiments may employ AARS polypeptides tomodulate systemic inflammation, such as to reduce or manage systemicinflammation. In certain embodiments, systemic inflammation may byassociated with systemic inflammatory response syndrome (SIRS), awhole-body inflammatory condition with a variety of potential causes.SIRS may be characterized or identified according to routine diagnostictechniques. As one non-limiting example, SIRS may be identified by thepresence of two or more of the following: (i) a body temperature that isless than 36° C. or greater than 38° C., (ii) a heart rate that isgreater than 90 beats per minute, (iii) tachypnea (high respiratoryrate), with greater than 20 breaths per minute; or, an arterial partialpressure of carbon dioxide less than 4.3 kPa (32 mmHg), and (iv) whiteblood cell count less than 4000 cells/mm³ (4×10⁹ cells/L) or greaterthan 12,000 cells/mm³ (12×10⁹ cells/L); or the presence of greater than10% immature neutrophils (band forms).

SIRS is broadly classified as either infectious or non-infectious. Mostgenerally, infectious SIRS is associated with sepsis, a whole-bodyinflammatory state combined with a known or suspected infection, whichincludes bacteremia, viremia, parasitemia, and toxic shock syndrome.Sepsis may be associated with a wide variety of infectious agents,including, without limitation, bacteria such as Streptococcusagalactiae, Escherichia coli, Haemophilus influenzae, Listeriamonocytogenes, Coagulase-negative Staphylococcus, Staphylococcus aureus,Klebsiella species, Pseudomonas aeruginosa, Enterobacter species, S.agalactiae, Serratia species, Acinetobacter species, Streptococcuspneumoniae, Salmonella species, and Neisseria meningitidis; viruses suchas rubella, cytomegalovirus, herpes simplex and the chickenpox virus;parasites such as in malarial infection (e.g., Plasmodium falciparum),trypanosomiasis, and filariasis; and fungi such as Candida species,Aspergillus species, Histoplasma species, Cryptococcus neoformans,Coccidioides immitis, Blastomyces dermatitidis, and Pneumocystiscarinii. In certain instances, infections in the lungs (e.g.,pneumonia), bladder and kidneys (e.g., urinary tract infections), skin(e.g., cellulitis), abdomen (e.g., appendicitis), and other areas (e.g.,meningitis) can spread and lead to sepsis AARS polypeptides may be usedto modulate inflammation associated with any of these infectious agents,whether sepsis is present or otherwise.

Noninfectious SIRS may be associated with trauma, burns, pancreatitis,ischemia, hemorrhage, surgical complications, adrenal insufficiency,pulmonary embolism, aortic aneurysm, cardiac tamponade, anaphylaxis, anddrug overdose, among others. SIRS is often complicated by the failure ofone or more organs or organ system, including those described herein.Specific examples include acute lung injury, acute kidney injury, shock,and multiple organ dysfunction syndrome, among others. Typically, SIRSis treated by focusing on the underlying problem (e.g., adequate fluidreplacement for hypovolemia, IVF/NPO for pancreatitis,epinephrine/steroids/benadryl for anaphylaxis). In certain instances,selenium, glutamine, and eicosapentaenoic acid have shown effectivenessin improving symptoms of SIRS, and antioxidants such as vitamin E mayalso be helpful. Hence, AARS polypeptides may be used to treat or manageSIRS and the complications of SIRS, alone or in combination with othertherapies.

Systemic inflammation may also be associated with “cytokine storm,” adangerous immune reaction caused by a positive feedback loop betweencytokines and immune cells, resulting in highly elevated levels ofvarious cytokines. In certain instances, cytokine storm(hypercytokinemia) includes the systemic release of numerous knowninflammatory mediators such as cytokines, oxygen free radicals, andcoagulation factors). Included are elevated levels of pro-inflammatorycytokines such as TNF-alpha, IL-1, and IL-6, and anti-inflammatorycytokines such as IL-10 and IL-1 receptor antagonist. Cytokine stormscan occur in a number of infectious and non-infectious diseasesincluding graft versus host disease (GVHD), acute respiratory distresssyndrome (ARDS), sepsis, avian influenza, smallpox, and SIRS. Cytokinestorm may also be induced by certain medications. Treatment includesOX40 IG, which reduces T-cell responses, ACE inhibitors, Angiotensin IIreceptor blockers, corticosteroids, gemfibrozil, free radicalscavengers, and TNF-α blockers. Accordingly, AARS polypeptides may beemployed to treat or manage cytokine storm, alone or in combination withother therapies.

Certain embodiments may employ AARS polypeptides to reduce any one ormore of granulomatous inflammation, fibrinous inflammation, purulentinflammation, serous inflammation, or ulcerative inflammation.Granulomatous inflammation is characterized by the formation ofgranulomas, typically resulting from a response to infectious agentssuch as tuberculosis, leprosy, and syphilis. Fibrinous inflammationresults from a large increase in vascular permeability, which allowsfibrin to pass through the blood vessels. If an appropriatepro-coagulative stimulus is present, such as a cancer cell, a fibrinousexudate is deposited. This process is commonly seen in serous cavities,where the conversion of fibrinous exudate into a scar can occur betweenserous membranes, limiting their function. Purulent inflammation resultsfrom the formation of a large amount of pus, which consists ofneutrophils, dead cells, and fluid. Infection by pyogenic bacteria suchas staphylococci is characteristic of this kind of inflammation. Large,localized collections of pus enclosed by surrounding tissues are calledabscesses. Serous inflammation is characterized by the copious effusionof non-viscous serous fluid, commonly produced by mesothelial cells ofserous membranes, but may also be derived from blood plasma. Examples ofthis type of inflammation include skin blisters. Ulcerativeinflammation, which typically occurs near an epithelium, results in thenecrotic loss of tissue from the surface, thereby exposing lower layersof tissue. The subsequent excavation of the epithelium is known as anulcer.

AARS polypeptides may also be employed in the treatment of physicalinjuries or wounds. Examples abrasions, bruises, cuts, puncture wounds,lacerations, impact wounds, concussions, contusions, thermal burns,frostbite, chemical burns, sunburns, gangrene, necrosis, desiccations,radiation burns, radioactivity burns, smoke inhalation, torn muscles,pulled muscles, torn tendons, pulled tendons, pulled ligaments, tornligaments, hyperextensions, torn cartilage, bone fractures, pinchednerves, ulcers, and gunshot or other traumatic wounds.

AARS polypeptides may also be employed to treat or manage idiopathicinflammation or inflammation of unknown etiology. Also included arecombination therapies, in which one or more AARS polypeptides areadministered or utilized in combination with one or more other therapiesfor any of the inflammatory diseases or conditions described herein,including those therapies that are commonly available and known in theart. Examples of combination therapies include the use of standardanti-inflammatory agents such as non-steroidal anti-inflammatory drugs(NSAIDs), immune selective anti-inflammatory derivatives (ImSAIDs), andsteroids (e.g., corticosteroids), anti-infectives such as antibioticsand anti-viral agents, anti-oxidants, cytokines, chemotherapeutic agentsand other anti-cancer therapies, and immunosuppressive therapies.

Criteria for assessing the signs and symptoms of inflammatory and otherconditions, including for purposes of making differential diagnosis andalso for monitoring treatments such as determining whether atherapeutically effective dose has been administered in the course oftreatment, e.g., by determining improvement according to acceptedclinical criteria, will be apparent to those skilled in the art and areexemplified by the teachings of e.g., Berkow et al., eds., The MerckManual, 16^(th) edition, Merck and Co., Rahway, N.J., 1992; Goodman etal., eds., Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 10^(th) edition, Pergamon Press, Inc., Elmsford, N.Y.,(2001); Avery's Drug Treatment: Principles and Practice of ClinicalPharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd., Williamsand Wilkins, Baltimore, Md. (1987); Ebadi, Pharmacology, Little, Brownand Co., Boston, (1985); Osolci al., eds., Remington's PharmaceuticalSciences, 18^(th) edition, Mack Publishing Co., Easton, Pa. (1990);Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk,Conn. (1992).

Certain embodiments may employ AARS polypeptides to increaseinflammation. For instance, depending on the needs of the subject,certain embodiments may increase acute inflammation or increase acuteinflammatory responses or both. Certain embodiments may increase chronicinflammation or chronic inflammatory responses or both. Certainembodiments may increase both acute and chronic inflammation. Certainembodiments may increase local or systemic inflammation or both.

In certain embodiments, AARS polypeptides may be used to treat or manageimmunodeficiencies, including primary immunodeficiencies and secondaryimmunodeficiencies, in which the body may not mount an adequateinflammatory response. Examples of primary immunodeficiencies includevarious autosomal recessive and X-linked genetic conditions such asT-cell and B-cell immunodeficiencies, including combined T-cell andB-cell immunodeficiencies, antibody deficiencies, well-definedsyndromes, immune dysregulation diseases, phagocyte disorders, innateimmunity disorders, and complement deficiencies.

Examples of T-cell and B-cell immunodeficiencies include T-/B+deficiencies such as γc deficiency, JAK3 deficiency, interleukin 7receptor chain α deficiency, CD45 deficiency, CD3δ/CD3ε deficiency; andT-/B− deficiencies such as RAG 1/2 deficiency, DCLRE1C deficiency,adenosine deaminase (ADA) deficiency, reticular dysgenesis. Additionalexamples include Omenn syndrome, DNA ligase type IV deficiency, CD40ligand deficiency, CD40 deficiency, purine nucleoside phosphorylase(PNP) deficiency, MHC class II deficiency, CD3γ deficiency, CD8deficiency, ZAP-70 deficiency, TAP-1/2 deficiency, and winged helixdeficiency.

Examples of antibody deficiencies include X-linked agammaglobulinemia(btk deficiency, or Bruton's agammaglobulinemia), μ-Heavy chaindeficiency, 1-5 deficiency, Igα deficiency, BLNK deficiency, thymomawith immunodeficiency, common variable immunodeficiency (CVID), ICOSdeficiency, CD19 deficiency, TACI (TNFRSF13B) deficiency, and BAFFreceptor deficiency. Additional examples include AID deficiency, UNGdeficiency, heavy chain deletions, kappa chain deficiency, isolated IgGsubclass deficiency, IgA with IgG subclass deficiency, selectiveimmunoglobulin A deficiency, and transient hypogammaglobulinemia ofinfancy (THI).

Examples of “well-defined syndromes” include Wiskott-Aldrich syndrome,ataxia telangiectasia, ataxia-like syndrome, Nijmegen breakage syndrome,Bloom syndrome, DiGeorge syndrome, immuno-osseous dysplasias such ascartilage-hair hypoplasia, Schimke syndrome, Hermansky-Pudlak syndrometype 2, Hyper-IgE syndrome, chronic mucocutaneous candidiasis.

Examples of immune dysregulation diseases include immunodeficiency withhypopigmentation or albinism such as Chediak-Higashi syndrome andGriscelli syndrome type 2, familial hemophagocytic lymphohistiocytosissuch as perforin deficiency, MUNC13D deficiency, and syntaxin 11deficiency, X-linked lymphoproliferative syndrome, autoimmunelymphoproliferative syndrome such as type 1a (CD95 defects), type 1b(Fas ligand defects), type 2a (CASP10 defects), and type 2b (CASP8defects), autoimmune polyendocrinopathy with candidiasis and ectodermaldystrophy, and immunodysregulation polyendocrinopathy enteropathyX-linked syndrome. Additionally, diseases affecting the bone marrow mayresult in abnormal or few leukocytes, such as leukopenia. Leukopenia canbe induced by certain infections and diseases, including viralinfection, Rickettsia infection, some protozoa, tuberculosis, andcertain cancers

Examples of phagocyte disorders include severe congenital neutropeniasuch as ELA2 deficiency (e.g., with myelodysplasia), GFI1 deficiency(with T/B lymphopenia) or G-CSFR deficiency (G-CSF-unresponsive),Kostmann syndrome, cyclic neutropenia, X-linkedneutropenia/myelodysplasia, leukocyte adhesion deficiency types 1, 2 and3, RAC2 deficiency, β-actin deficiency, localized juvenileperiodontitis, Papillon-Lefèvre syndrome, specific granule deficiency,Shwachman-Diamond syndrome, chronic granulomatous disease, includingX-linked and autosomal forms, neutrophil glucose-6-phosphatedehydrogenase deficiency, IL-12 and IL-23 β1 chain deficiency, IL-12p40deficiency, interferon γ receptor 1 deficiency, interferon γ receptor 2deficiency, and STAT1 deficiency.

Examples of innate immunity deficiencies include hypohidrotic ectodermaldysplasia such as NEMO deficiency and IKBA deficiency, IRAK-4deficiency, WHIM syndrome (warts, hypogammaglobulinaemia, infections,myleokathexis), and epidermodysplasia verruciformis. Examples ofcomplement deficiencies and examplary associated conditions include C1qdeficiency (e.g., lupus-like syndrome, rheumatoid disease, infections),C1r deficiency, C4 deficiency, C2 deficiency (e.g., lupus-like syndrome,vasculitis, polymyositis, pyogenic infections), C3 deficiency (e.g.,recurrent pyogenic infections), C5 deficiency (e.g., neisserialinfections), C6 deficiency, C7 deficiency (e.g., vasculitis), C8a andC8b deficiency, C9 deficiency (e.g., neisserial infections),C1-inhibitor deficiency (e.g., hereditary angioedema), Factor Ideficiency (pyogenic infections), Factor H deficiency (e.g.,haemolytic-uraemic syndrome, membranoproliferative glomerulonephritis),Factor D deficiency (e.g., neisserial infections), Properdin deficiency(e.g., neisserial infections), MBP deficiency (e.g., pyogenicinfections), and MASP2 deficiency.

Primary immune deficiencies can be diagnosed according to routinetechniques in the art. Exemplary diagnostic tests include, withoutlimitation, performing counts of the different types of mononuclearcells in the blood (e.g., lymphocytes and monocytes, includinglymphocytes, different groups of B lymphocytes such as CD19+, CD20+, andCD21+ lymphocytes, natural killer cells, and monocytes positive forCD15+), measuring the presence of activation markers (e.g., HLA-DR,CD25, CD80), performing tests for T cell function such as skin tests fordelayed-type hypersensitivity, cell responses to mitogens and allogeneiccells, cytokine production by cells, performing tests for B cellfunction such as by identifying antibodies to routine immunizations andcommonly acquired infections and by quantifying IgG subclasses, andperforming tests or phagocyte function, such as by measuring thereduction of nitro blue tetrazolium chloride, and performing assays ofchemotaxis and bactericidal activity. AARS polypeptides may therefore beused to stimulate or maintain acute inflammation or acute inflammatoryresponses in subjects with a primary immunodeficiency, as describedherein and known in the art.

Examples of causes of secondary immunodeficiencies include malnutrition,aging, and medications (e.g., chemotherapy, disease-modifyinganti-rheumatic drugs, immunosuppressive drugs after organ transplants,glucocorticoids). Additional causes include various cancers, includingcancers of the bone marrow and blood cells (e.g., leukemia, lymphoma,multiple myeloma), and certain chronic infections, such as acquiredimmunodeficiency syndrome (AIDS), caused by the human immunodeficiencyvirus (HIV). AARS polypeptides may be used to stimulate or maintainacute inflammation or acute inflammatory responses in subjects with animmunodeficiency, as described herein and known in the art. AARSpolypeptides may also be used to stimulate or maintain chronicinflammation or chronic inflammatory responses in subjects with asecondary immunodeficiency, as described herein and known in the art.

In certain embodiments, for example, methods are provided for modulatingtherapeutically relevant cellular activities including, but not limitedto, cellular metabolism, cell differentiation, cell proliferation, celldeath, cell mobilization, cell migration, gene transcription, mRNAtranslation, cell impedance, cytokine production, and the like,comprising contacting a cell with an AARS composition as describedherein. In certain embodiments, the AARS polypeptides (e.g., QRSpolypeptides) or compositions thereof modulate the cytokine response ofcells to immune-stimulating antigens, including autoimmunedisorder-related antigens and foreign antigens such aslipopolysaccharide (LPS). In certain embodiments, the AARS polypeptides(e.g., QRS polypeptides) or compositions thereof inhibit the cytokineresponse of cells to immune-stimulating antigens, as above. In certainembodiments, the cells are peripheral blood mononuclear cells (PBMCs).

In certain particular embodiments, AARS polypeptides (e.g., QRSpolypeptides) or compositions thereof are provided for inhibiting TNF-αproduction or secretion in mammalian cells, such as PBMCs, either invivo or in vitro. In certain particular embodiments, QRS polypeptides orcompositions thereof are provided for inhibiting IL-12 production orsecretion in mammalian cells, such as PBMCs, either in vivo or in vitro.In certain embodiments, QRS polypeptides inhibit the TNF-α orIL-12-based secretion response of cells to immune-stimulating antigens,including autoimmune disorder-related antigens and foreign antigens suchas lipopolysaccharide (LPS). Accordingly, the AARS polypeptides (e.g.,QRS polypeptides) may be employed in treating essentially any cell ortissue or subject that would benefit from modulation of one or more suchactivities.

The AARS polypeptides (e.g., QRS polypeptides) and compositions may alsobe used in any of a number of therapeutic contexts including, forexample, those relating to the treatment or prevention of neoplasticdiseases, immune system diseases (e.g., autoimmune diseases andinflammation), infectious diseases, metabolic diseases,neuronal/neurological diseases, muscular/cardiovascular diseases,diseases associated with aberrant hematopoiesis, diseases associatedwith aberrant angiogenesis, diseases associated with aberrant cellsurvival, and others.

For example, in certain illustrative embodiments, the AARS polypeptides(e.g., QRS polypeptides) and compositions of the invention may be usedto modulate angiogenesis, e.g., via modulation of endothelial cellproliferation and/or signaling. Endothelial cell proliferation and/orcell signaling may be monitored using an appropriate cell line (e.g.,Human microvascular endothelial lung cells (HMVEC-L) and Human umbilicalvein endothelial cells (HUVEC)), and using an appropriate assay (e.g.,endothelial cell migration assays, endothelial cell proliferationassays, tube-forming assays, matrigel plug assays, etc.), many of whichare known and available in the art.

Therefore, in related embodiments, the compositions of the invention maybe employed in the treatment of essentially any cell or tissue orsubject that would benefit from modulation of angiogenesis. For example,in some embodiments, a cell or tissue or subject experiencing orsusceptible to angiogenesis (e.g., an angiogenic condition) may becontacted with a suitable composition of the invention to inhibit anangiogenic condition. In other embodiments, a cell or tissueexperiencing or susceptible to insufficient angiogenesis (e.g., anangiostatic condition) may be contacted with an appropriate compositionof the invention in order to interfere with angiostatic activity and/orpromote angiogenesis.

Illustrative examples of angiogenic conditions include, but are notlimited to, age-related macular degeneration (AMD), cancer (both solidand hematologic), developmental abnormalities (organogenesis), diabeticblindness, endometriosis, ocular neovascularization, psoriasis,rheumatoid arthritis (RA), and skin disclolorations (e.g., hemangioma,nevus flammeus or nevus simplex). Examples of anti-angiogenic conditionsinclude, but are not limited to, cardiovascular disease, restenosis,tissue damage after reperfusion of ischemic tissue or cardiac failure,chronic inflammation and wound healing.

The compositions of the invention may also be useful as immunomodulatorsfor treating anti- or pro-inflammatory indications by modulating thecells that mediate, either directly or indirectly, autoimmune and/orinflammatory disease, conditions and disorders. The utility of thecompositions of the invention as immunomodulators can be monitored usingany of a number of known and available techniques in the art including,for example, migration assays (e.g., using leukocytes or lymphocytes),cytokine production assays (e.g., TNF-α, IL-12), or cell viabilityassays (e.g., using B-cells, T-cells, monocytes or NK cells).

Illustrative immune system diseases, disorders or conditions that may betreated according to the present invention include, but are not limitedto, primary immunodeficiencies, immune-mediated thrombocytopenia,Kawasaki syndrome, bone marrow transplant (for example, recent bonemarrow transplant in adults or children), chronic B cell lymphocyticleukemia, HIV infection (for example, adult or pediatric HIV infection),chronic inflammatory demyelinating polyneuropathy, post-transfusionpurpura, and the like.

Additionally, further diseases, disorders and conditions includeGuillain-Barre syndrome, anemia (for example, anemia associated withparvovirus B19, patients with stable multiple myeloma who are at highrisk for infection (for example, recurrent infection), autoimmunehemolytic anemia (for example, warm-type autoimmune hemolytic anemia),thrombocytopenia (for example, neonatal thrombocytopenia), andimmune-mediated neutropenia), transplantation (for example,cytomegalovirus (CMV)-negative recipients of CMV-positive organs),hypogammaglobulinemia (for example, hypogammaglobulinemic neonates withrisk factor for infection or morbidity), epilepsy (for example,intractable epilepsy), systemic vasculitic syndromes, myasthenia gravis(for example, decompensation in myasthenia gravis), dermatomyositis, andpolymyositis.

Further autoimmune diseases, disorders and conditions include but arenot limited to, autoimmune hemolytic anemia, autoimmune neonatalthrombocytopenia, idiopathic thrombocytopenia purpura,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, glomerulonephritis (forexample, IgA nephropathy), multiple sclerosis, neuritis, uveitisophthalmia, polyendocrinopathies, purpura (for example,Henloch-Scoenlein purpura), Reiter's disease, stiff-man syndrome,autoimmune pulmonary inflammation, Guillain-Barre Syndrome, insulindependent diabetes mellitus, and autoimmune inflammatory eye disease.

Additional autoimmune diseases, disorders or conditions include, but arenot limited to, autoimmune thyroiditis; hypothyroidism, includingHashimoto's thyroiditis and thyroiditis characterized, for example, bycell-mediated and humoral thyroid cytotoxicity; SLE (which is oftencharacterized, for example, by circulating and locally generated immunecomplexes); Goodpasture's syndrome (which is often characterized, forexample, by anti-basement membrane antibodies); pemphigus (which isoften characterized, for example, by epidermal acantholytic antibodies);receptor autoimmunities such as, for example, Graves' disease (which isoften characterized, for example, by antibodies to a thyroid stimulatinghormone receptor; myasthenia gravis, which is often characterized, forexample, by acetylcholine receptor antibodies); insulin resistance(which is often characterized, for example, by insulin receptorantibodies); autoimmune hemolytic anemia (which is often characterized,for example, by phagocytosis of antibody-sensitized red blood cells);and autoimmune thrombocytopenic purpura (which is often characterized,for example, by phagocytosis of antibody-sensitized platelets).

Further autoimmune diseases, disorders or conditions include, but arenot limited to, rheumatoid arthritis (which is often characterized, forexample, by immune complexes in joints); scleroderma with anti-collagenantibodies (which is often characterized, for example, by nucleolar andother nuclear antibodies); mixed connective tissue disease, (which isoften characterized, for example, by antibodies to extractable nuclearantigens, for example, ribonucleoprotein); polymyositis/dermatomyositis(which is often characterized, for example, by nonhistone anti-nuclearantibodies); pernicious anemia (which is often characterized, forexample, by antiparietal cell, antimicrosome, and anti-intrinsic factorantibodies); idiopathic Addison's disease (which is often characterized,for example, by humoral and cell-mediated adrenal cytotoxicity);infertility (which is often characterized, for example, byantispennatozoal antibodies); glomerulonephritis (which is oftencharacterized, for example, by glomerular basement membrane antibodiesor immune complexes); by primary glomerulonephritis, by IgA nephropathy;bullous pemphigoid (which is often characterized, for example, by IgGand complement in the basement membrane); Sjogren's syndrome (which isoften characterized, for example, by multiple tissue antibodies and/orthe specific nonhistone antinuclear antibody (SS-B)); diabetes mellitus(which is often characterized, for example, by cell-mediated and humoralislet cell antibodies); and adrenergic drug resistance, includingadrenergic drug resistance with asthma or cystic fibrosis (which isoften characterized, for example, by beta-adrenergic receptorantibodies).

Still further autoimmune diseases, disorders or conditions include, butare not limited to chronic active hepatitis (which is oftencharacterized, for example by smooth muscle antibodies); primary biliarycirrhosis (which is often characterized, for example, byanti-mitochondrial antibodies); other endocrine gland failure (which ischaracterized, for example, by specific tissue antibodies in somecases); vitiligo (which is often characterized, for example, byanti-melanocyte antibodies); vasculitis (which is often characterized,for example, by immunoglobulin and complement in vessel walls and/or lowserum complement); post-myocardial infarction conditions (which areoften characterized, for example, by anti-myocardial antibodies);cardiotomy syndrome (which is often characterized, for example, byanti-myocardial antibodies); urticaria (which is often characterized,for example, by IgG and IgM antibodies to IgE); atopic dermatitis (whichis often characterized, for example, by IgG and IgM antibodies to IgE);asthma (which is often characterized, for example, by IgG and IgMantibodies to IgE); inflammatory myopathies; and other inflammatory,granulomatous, degenerative, and atrophic disorders.

In other embodiments, the AARS polypeptides (e.g., QRS polypeptides) andcompositions of the invention may be used to modulate cellularproliferation and/or survival and, accordingly, for treating orpreventing diseases, disorders or conditions characterized byabnormalities in cellular proliferation and/or survival. For example, incertain embodiments, the QRS compositions may be used to modulateapoptosis and/or to treat diseases or conditions associated withabnormal apoptosis. Apoptosis is the term used to describe the cellsignaling cascade known as programmed cell death. Various therapeuticindications exist for molecules that induce apoptosis (e.g. cancer), aswell as those that inhibit apoptosis (i.e. stroke, myocardialinfarction, sepsis, etc.). Apoptosis can be monitored by any of a numberof available techniques known and available in the art including, forexample, assays that measure fragmentation of DNA, alterations inmembrane asymmetry, activation of apoptotic caspases and/or release ofcytochrome C and AIF.

Illustrative diseases associated with increased cell survival, or theinhibition of apoptosis include, but are not limited to, cancers (suchas follicular lymphomas, carcinomas, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Graves' disease, Hashimoto's thyroiditis,autoimmune diabetes, biliary cirrhosis, Behçet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenicpurpura, and rheumatoid arthritis) and viral infections (such as herpesviruses, pox viruses and adenoviruses), inflammation, graft vs. hostdisease (acute and/or chronic), acute graft rejection, and chronic graftrejection.

Further illustrative diseases or conditions associated with increasedcell survival include, but are not limited to, progression and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (for example, acute lymphocytic leukemia,acute myelocytic leukemia, including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(for example, chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia), myelodysplastic syndrome polycythemia vera,lymphomas (for example, Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Illustrative diseases associated with increased apoptosis include, butare not limited to, AIDS (such as HIV-induced nephropathy and HIVencephalitis), neurodegenerative disorders (such as Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, cerebellar degeneration and brain tumor or prior associateddisease), autoimmune disorders such as multiple sclerosis, Sjogren'ssyndrome, Graves' disease, Hashimoto's thyroiditis, autoimmune diabetes,biliary cirrhosis, Behçet's disease, Crohn's disease, polymyositis,systemic lupus erythematosus, immune-related glomerulonephritis,autoimmune gastritis, thrombocytopenic purpura, and rheumatoidarthritis, myelodysplastic syndromes (such as aplastic anemia), graftvs. host disease (acute and/or chronic), ischemic injury (such as thatcaused by myocardial infarction, stroke and reperfusion injury), liverinjury or disease (for example, hepatitis related liver injury,cirrhosis, ischemia/reperfusion injury, cholestosis (bile duct injury)and liver cancer), toxin-induced liver disease (such as that caused byalcohol), septic shock, ulcerative colitis, cachexia, and anorexia.

In still further embodiments, the compositions of the invention may beused in the treatment of neuronal/neurological diseases or disorders,illustrative examples of which include Parkinson's disease, Alzheimer'sdisease, Pick's disease, Creutzfeldt-Jacob disease, Huntington's chorea,alternating hemiplegia, amyotrophic lateral sclerosis, ataxia, cerebralpalsy, chronic fatigue syndrome, chronic pain syndromes, congenitalneurological anomalies, cranial nerve diseases, delirium, dementia,demyelinating diseases, dysautonomia, epilepsy, headaches, Huntington'sdisease, hydrocephalus, meningitis, movement disorders, muscle diseases,nervous system neoplasms, neurocutaneous syndromes, neurodegenerativediseases, neurotoxicity syndromes, ocular motility disorders, peripheralnervous system disorders, pituitary disorders, porencephaly, Rettsyndrome, sleep disorders, spinal cord disorders, stroke, sydenham'schorea, tourette syndrome, nervous system trauma and injuries, etc.

Furthermore, additional embodiments relate to the use of thecompositions of the invention in the treatment of metabolic disorderssuch as adrenoleukodystrophy, Krabbe's disease (globoid cellleukodystrophy), metachromatic leukodystrophy, Alexander's disease,Canavan's disease (spongiform leukodystrophy), Pelizaeus-Merzbacherdisease, Cockayne's syndrome, Hurler's disease, Lowe's syndrome, Leigh'sdisease, Wilson's disease, Hallervorden-Spatz disease, Tay-Sachsdisease, etc. The utility of the compositions of the invention inmodulating metabolic processes may be monitored using any of a varietyof techniques known and available in the art including, for example,assays which measure adipocyte lipogenesis or adipocyte lipolysis.

In more specific embodiments of the invention, the AARS polypeptides(e.g., QRS polypeptides) and compositions of the invention may be usedto modulate cellular signaling, for example, via cell signaling proteins(e.g., Akt). Cell signaling may be monitored using any of a number ofwell known assays. For example, the induction of general cell signalingevents can be monitored through altered phosphorylation patterns of avariety of target proteins. Detection of cell signaling activities inresponse to treatment of cells with QRS polypeptides therefore serves asan indicator of distinct biological effects. Target proteins used forthis assay may be selected so as to encompass key components of majorcellular signaling cascades, thereby providing a broad picture of thecell signaling landscape and its therapeutic relevance. Generally, suchassays involve cell treatment with QRS polypeptides followed byimmunodetection with antibodies that specifically detect thephosphorylated (activated) forms of the target proteins.

Illustrative target proteins used for monitoring therapeuticallyrelevant cell signaling events may include, but are not limited to: p38MAPK (mitogen-activated protein kinase; activated by cellular stress andinflammatory cytokines; involved in cell differentiation and apoptosis);SAPK/JNK (stress-activated protein kinase/Jun-amino-terminal kinase;activated by cellular stresses and inflammatory cytokines); Erk1/2,p44/42 MAPK (mitogen-activated protein kinase Erk1 and Erk2; activatedby wide variety of extracellular signals; involved in regulation of cellgrowth and differentiation); and Akt (activated by insulin and variousgrowth or survival factors; involved in inhibition of apoptosis,regulation of glycogen synthesis, cell cycle regulation and cellgrowth). General phosphorylation of tyrosine residues may also bemonitored as a general indicator of changes in cell signaling mediatedby phosphorylation.

Of course, it will be recognized that other classes of proteins, such ascell adhesion molecules (e.g., cadherins, integrins, claudins, catenins,selectins, etc.) and/or ion channel proteins may also be assayed formonitoring cellular events or activities modulated by the compositionsof the invention.

In other specific embodiments of the invention, the AARS polypeptides(e.g., QRS polypeptides) and compositions of the invention may be usedto modulate cytokine production by cells, for example, by leukocytes.Cytokine production may be monitored using any of a number of assaysknown in the art (i.e., RT-PCR, ELISA, ELISpot, flow cytometry, etc.).Generally, such assays involve cell treatment with AARS polypeptides(e.g., QRS polypeptides) polypeptides followed by detection of cytokinemRNA or polypeptides to measure changes in cytokine production.Detection of increases and/or decreases in cytokine production inresponse to treatment of cells with AARS polypeptides (e.g., QRSpolypeptides) therefore serves as an indicator of distinct biologicaleffects. QRS polypeptides of the invention may induce, enhance, and/orinhibit an immune or inflammatory response by modulating cytokineproduction. For example, AARS polypeptides (e.g., QRS polypeptides)polypeptides and compositions of the invention may be used to alter acytokine profile (i.e., type 1 vs. type 2) in a subject. Illustrativecytokines that may measured for monitoring biological effects of the QRScompositions include, but are not limited to IL-1α, IL-1β, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, IL-23 TGF-β, TNF-α,IFN-α, IFN-β, IFN-γ, RANTES, MIP-1α, MIP-1β, MCP-1, GM-CSF, G-CSF, etc.

Generally, a therapeutically effective amount of polypeptide isadministered to a subject or patient. In particular embodiments, theamount of polypeptide administered will typically be in the range ofabout 0.1 μg/kg to about 0.1 mg/kg to about 50 mg/kg of patient bodyweight. Depending on the type and severity of the disease, about 0.1μg/kg to about 0.1 mg/kg to about 50 mg/kg body weight (e.g., about0.1-15 mg/kg/dose) of polypeptide can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. For example, adosing regimen may comprise administering an initial loading dose ofabout 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg ofthe polypeptide, or about half of the loading dose. However, otherdosage regimens may be useful. A typical daily dosage might range fromabout 0.1 μg/kg to about 1 μg/kg to 100 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment is sustained untila desired suppression of disease symptoms occurs. The progress of theseand other therapies (e.g., ex vivo therapies) can be readily monitoredby conventional methods and assays and based on criteria known to thephysician or other persons of skill in the art.

Formulations and Pharmaceutical Compositions

The compositions of the invention comprise aminoacyl-tRNA synthetasepolypeptides, including truncations and/or variants thereof, formulatedin pharmaceutically-acceptable or physiologically-acceptable solutionsfor administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy. It will alsobe understood that, if desired, the compositions of the invention may beadministered in combination with other agents as well, such as, e.g.,other proteins or polypeptides or various pharmaceutically-activeagents. There is virtually no limit to other components that may also beincluded in the compositions, provided that the additional agents do notadversely affect the inflammatory response-modulating activities orother effects desired to be achieved.

In the pharmaceutical compositions of the invention, formulation ofpharmaceutically-acceptable excipients and carrier solutions iswell-known to those of skill in the art, as is the development ofsuitable dosing and treatment regimens for using the particularcompositions described herein in a variety of treatment regimens,including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to a subject. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as described,for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 andU.S. Pat. No. 5,399,363 (each specifically incorporated herein byreference in its entirety). Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form should be sterileand should be fluid to the extent that easy syringability exists. Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent with thevarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, polynucleotides, and peptidecompositions directly to the lungs via nasal aerosol sprays have beendescribed e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212(each specifically incorporated herein by reference in its entirety).Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

Also included are topical formulations. Examples of topical formulationsinclude creams, ointments, pastes, lotions, and gels.

In certain embodiments, the delivery may occur by use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticleor the like. The formulation and use of such delivery vehicles can becarried out using known and conventional techniques.

All publications, patent applications, and issued patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or issued patent were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of noncritical parameters thatcould be changed or modified to yield essentially similar results.

EXAMPLES Example 1 Amino Acyl-tRNA Synthetase Polypeptides ReduceNeutrophil Migration and Infiltration into the Lungs afterLipopolysaccharide (LPS) Challenge

Neutrophil migration from the circulatory system to the lungs isimplicated in chronic pulmonary obstructive disease (COPD) (see, e.g.,R. A. Stockley, Chest 121:151S-155S, 2002). CXCR-2 expression can play arole in neutrophil migration (see, e.g., Rios-Santos et al., AmericanJournal of Respiratory and Critical Care Medicine 175:490-497, 2007). Todetermine whether tyrosyl-tRNA synthetase (YRS) polypeptides andhistidyl-tRNA synthetase (HisRS) polypeptides can be used to treatneutrophil-mediated disorders, male C57BL/6 mice were anesthetized,injected intra-nasally with 50 μl of a 200 μg/ml lipopolysaccharide(LPS, Sigma-Aldrich Cat# L2880) and sacrificed approximately 8 hoursafter LPS administration. Prior to exposure to LPS, mice were treatedwith YRS polypeptides, HisRS polypeptides, or control. A trachealcatheter was inserted to collect bronchoalveolar lavage (BAL) samples byflushing the lungs five times with 1 ml of ice-cold saline solution.Lavage fluid was collected for later cell staining and counting.

As shown in FIG. 1A, neutrophils are typically absent from the BAL fluidrecovered from healthy, untreated animals. Intra-nasal LPSadministration resulted in the infiltration of circulating neutrophilsinto the lungs and in a marked increase in BAL neutrophils (FIG. 1A, LPSgroup). Intraperitoneal pretreatment with dexamethasone, a syntheticcorticosteroid used as positive control in this experiment, resulted ina diminished ability of neutrophils to relocate to the lungs after LPSchallenge (FIG. 1A, Dex group). Similarly, intravenous administration oftwo doses of the YRS and HisRS synthetase polypeptides at 7-8 hours and1.5 hour prior to LPS administration, respectively, resulted in adrastic reduction in BAL neutrophils. The results for YRS are shown inFIG. 1 (FIG. 1A, Y341A group; and FIG. 1B, Mini-YRS group). Full-lengthHisRS polypeptide exerted similar effects on neutrophils (FIG. 2A), andwas also capable of decreasing eosinophil migration to the lungs (FIG.2B). Similar results are seen for tryptophanyl-tRNA synthetase (WRS)polypeptides.

Example 2 Tyrosyl-tRNA Synthetase Polypeptides Stimulate Migration of293 and CHO Cell Lines Transfected with the CXCR-2 Receptor

The effects of tyrosyl-tRNA synthetase polypeptides on CXCR-2 signalingwas tested by measuring the migration of CXCR-2 expressing cells inresponse to said polypeptides. 293/CXCR-2 cells were maintained in DMEMmedium supplemented with 10% heat-inactivated FBS, 1%Penicillin-Streptomycin and 800 μg/ml Geneticin, all purchased fromInvitrogen, Carlsbad, Calif. DMEM medium with 0.1% BSA was used asmigration buffer. Prior to migration assay, cells were serum-starved for30 minutes in migration buffer, centrifuged at 200 g for 5 minutes andresuspended in migration buffer at a final density of 1×10⁶ cells/ml.100 μl were added to 6.5 mm transwell filter inserts (Costar, Cambridge,Mass.) and 600 μl migration buffer containing a control chemokine, thetyrosyl-tRNA synthetase polypeptides or buffer only were added to theplate lower chambers. Cells were allowed to migrate for 4 hours and theremaining cells in the upper chamber (transwell filter inserts) wereremoved with a cotton swap. The filter inserts were then transferred toa new 24-well plate containing 500 μl cell dissociation buffer(Invitrogen, Carlsbad, Calif.) and 12 μg/ml Calcein AM (Invitrogen,Carlsbad, Calif.). After 1 hour incubation at 37° C., cells werecollected and resuspended in 100 μl PBS, transferred into a 384-wellopaque Greiner plate, and counted by fluorescence in a plate reader.

CHO-K1/CXCR-2 cells were maintained in F12 medium supplemented with 10%heat-inactivated FBS, 1% Penicillin-Streptomycin-Glutamine and 800 μg/mlGeneticin. F12 medium with 0.5% BSA was used as migration buffer. Priorto migration, cells were serum-starved for 30 minutes in migrationbuffer, collected by using cell dissociation buffer, spun down at 200 gfor 5 minutes and resuspended in migration buffer at the final densityof 1×10⁶ cells/ml. 100 μl were added to 6.5 mm transwell filter insertsand 600 μl migration buffer containing a control chemokine, thetyrosyl-tRNA synthetase polypeptides or buffer only were added to theplate lower chambers. Cells were allowed to migrate for 3 hours and theremaining cells in the upper chamber (transwell filter inserts) wereremoved with a cotton swap. The filter inserts were then transferred toa new 24-well plate containing 500 μl PBS and 12 μg/ml Calcein AM. After30 minutes incubation at 37° C., filters were transferred again into anew 24-well plate containing 500 μl phenol/red-free trypsin. After 2 to5 minutes incubation, detached cells were collected and resuspended in100 μl PBS, transferred into a 384 well opaque Greiner plate and countedby fluorescence in a plate reader.

FIG. 3 shows the ability of the tyrosyl-tRNA synthetase polypeptides toinduce migration of CXCR-2 transfected cells.

Example 3 Tyrosyl-tRNA Synthetase Polypeptides StimulatePolymorphonuclear (PMN) Cell Migration

To test the effects of YRS polypeptides on PMN cell migration, humangranulocyte cells were purified from fresh human peripheral blood usingRosetteSep® Human Granulocyte Enrichment Kit (StemCell Technologies,Vancouver, BC) according to the manufacturer's instructions. Serum-freeRPMI medium supplemented with 0.5% FBS was used as migration buffer.4×10⁷ cells were resuspended in 1 ml migration buffer and incubated for30 minutes with 8 μl of a 1 mg/ml Calcein AM solution (Invitrogen,Carlsbad, Calif.). Cells were collected, spun down at 200 g for 5minutes without brake, washed once with migration buffer and resuspendedin the same buffer at a final density of 1×10⁷/ml.

100 μl were added to 6.5 mm transwell filter inserts (Costar, Cambridge,Mass.) and 600 μl migration buffer containing a control chemokine, thetyrosyl-tRNA synthetase polypeptides or buffer only were added to theplate lower chambers. Cells were allowed to migrate for 45 minutes inthe incubator and cells that migrated to the lower chamber werecollected, resuspended in 100 μl PBS, transferred into a 384-well opaqueGreiner plate and counted by fluorescence in a plate reader.

FIG. 4 shows the bell-shaped migration curve typically observed withchemokines. The tyrosyl-tRNA synthetase polypeptides induced a biphasicmigration of PMN both at low pM and at higher μM concentrations.

Example 4 Aspartyl-tRNA Synthetase Polypeptide D1 Induces Secretion ofBoth Pro- and Anti-Inflammatory Cytokines

To probe the possible connection between the D1 fragment (residues1-154) of full-length AspRS and inflammation, recombinant protein wasinjected intravenously into healthy mice, and changes in inflammatorycytokines (both pro- and anti-inflammatory) secreted into thebloodstream were observed relative to vehicle controls. Serum washarvested 2 and 6 hours post-injection and TNF-α and IL-10 levels weremeasured by ELISA.

Upon examination at 2 hours post-injection of D1, an increased secretionof both pro-inflammatory cytokines (TNF-α, MIP-1b, IL-12(p40), KC,MIP-2), and IL-10, an anti-inflammatory cytokine, was observed (FIGS. 5Aand 5B). At 6 hours post-injection of D1, the pro-inflammatory cytokinescould no longer be detected, but the levels of IL-10 anti-inflammatoryin the serum continued to increase (see FIGS. 5A and B).

To confirm these results, peripheral blood mononuclear cells (PBMCs)representing a mixture of both monocytes and lymphocytes isolated fromhuman donors were exposed to the D1 protein in vitro (as well as thefull-length AspRS protein), and the media was tested for the secretionof either TNF-α or IL-10 in response to treatment. Similar to theeffects observed in vivo, treatment with D1 resulted in secretion ofboth TNF-α (after 4 hours treatment) and IL-10 (after 24 hourstreatment) from the mixed cell population (see FIGS. 5C and D).

Example 5 Splice Variants HRS-SV9 and HRS-SV11 Increase IL-2 Secretionin Activated T-Cells

When antigen is presented by antigen presenting cells (APC), theearliest detectable response of T cell activation is the secretion ofcytokines, such as IL-2. Through autocrine secretion, IL-2 triggers Tcells proliferation, thereby generating cells required to eliminateantigen. Thus, regulators of IL-2 secretion serve as immunomodulatorsfor T lymphocyte-mediated immune responses.

Leukemia Jurkat T cells (ATCC No: TIB-152) are widely used for T cellactivation research, using IL-2 expression and release as an indicationof activation. For T cell activation, Jurkat T cells were stimulated byphorbol esters (PMA) and ionomycin (IOM). IL-2 secretion into media wasevaluated by ELISA. As expected, PMA and ionomycin stimulated Jurkat Tcells to release IL-2 in a dose dependent manner.

As shown in FIG. 6, HRS-SV9 and HRS-SV11, when co-applied with PMA andIOM significantly increased IL-2 secretion. Thus, both HRS-SV9 andHRS-SV11 exhibited immunomodulatory activity.

Example 6 Splice Variant HRS-SV9 Stimulates TNF-Alpha Secretion in PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated from humanblood. The cells were resuspended in RPMI media with 10% FBS to 1×10⁶cells/mL. One million cells were treated for 24 hours with HRS-SV9 at6.25, 12.5, 25, 50, 100, and 250 nM. PBMCs were also treated withLipopolysaccharide (LPS) at 1 EU/mL, PBS, or 100 nM Negative ControlProtein 1 or 2. After 24 hours, cell supernatant was collected bycentrifugation at 2000×g for 10 min and evaluated in a TNF-α ELISA assay(R&D Systems; Cat. DTA00C).

As shown in FIG. 7, HRS-SV9 stimulated PBMCs to secrete TNF-α in a dosedependent manner. In contrast, cells treated with PBS or negativecontrol proteins secreted minimal or no TNF-α (PBS, Neg. Ctrl. 1 andNeg. Ctrl. 2). LPS, a known inducer of TNF-α secretion, gave rise to apositive signal at 1 EU/ml. Although a minimal amount of LPS was presentin the HRS-SV9 protein (˜0.11 EU/mL at 250 nM), the TNF-α signalobserved for HRS-SV9 is above that which maybe attributed to LPS. Thus,the results of this example demonstrate that HRS-SV9 acts as a modulatorof TNF-α secretion.

Example 7 Generation and Identification of Endogenous HumanGlutaminyl-tRNA Synthetase (QRS) Fragments

Full-length recombinant human QRS (SEQ ID NO:25) was expressed andpurified from E. coli using nickel IMAC chromatography. Endogenousproteolytic fragments were generated through the process of purificationand subsequently characterized using LC/MS/MS. Without wishing to bebound by any one theory, it is believed that these fragments areindicative of those that would be created in human cells through theprocess of natural proteolysis.

To identify the residues at which proteolysis occurs for human QRS, theproteins were separated by SDS-PAGE run in 4-12% MOPS, gel slicescontaining the fragments were excised and subjected to in-gel trypsindigestion followed LC/MS/MS analysis. This process allowed theidentification of both the portion of the full-length protein from whichthe fragments were generated and the non-trypsin cleavage sites thatcould be attributed to endogenous proteolytic cleavage. All proteinfragments identified represented the N-terminal portion of QRS. SeeTable 1 below, and FIG. 8 (A-C).

TABLE 1 Endogenous QRS proteolytic fragments Whole mass N-term. C-term.Non-tryptic (Da) boundary boundary peptide found Q1 22200 1 183 Q2 265001 220 DVVENGETADQTESL220 (SEQ ID NO: 26) Q3 29800 1 249 TPGYVVTPHT249(SEQ ID NO: 27) Q4 25000 1 181-293 (200) Q5 24000 1 181-293

QRS fragments closely matching those identified by LC/MS/MS in Table 1above were cloned into an E. coli protein expression vector forover-expression and purification. Proteins were purified using NickelIMAC chromatography and contaminants were removed using a Sartobind Qmembrane (Sartorius). See FIG. 9.

Example 8 N-Terminal Proteolytic Fragments of QRS Inhibit LPS-InducedTNF-Alpha Secretion from PBMCs

To measure the effects of QRS polypeptides on TNF-α secretion,peripheral blood mononuclear cells (PBMCs) were isolated from humanblood obtained from healthy donors and treated with QRS polypeptides.The cells were resuspended in RPMI media with 10% FBS to 1×10⁶ cells/mL.One million cells were pre-treated for 30 minutes with a dose responseof 63 nM, 125 nM, 250 nM and 500 nM (463 nM for Q3) of each Q fragment.After 30 minutes, lipopolysaccharide (LPS, 0.5 EU/mL) was added topretreated and untreated cells. After 24 h, cell supernatant wascollected by centrifugation at 2000×g for 10 minutes and evaluated in aTNF-α ELISA (R&D Systems; Cat. DTA00C) per kit directions.

As shown in FIG. 10, pretreatment with all four QRS fragments inhibitedthe amount of TNF-α released from PBMCs upon stimulation with 0.5 EU/mlLPS.

Example 9 N-Terminal Proteolytic Fragment of QRS Inhibits LPS-InducedTNF-Alpha Secretion from PBMCs at 4 and 24 Hours

To measure the longer term effects of QRS polypeptides on TNF-αsecretion, peripheral blood mononuclear cells (PBMCs) were isolated fromhuman blood obtained from healthy donors and treated with QRSpolypeptides. The cells were resuspended in RPMI media with 10% FBS to1×10⁶ cells/mL. One million cells were pre-treated for 30 minutes with500 nM Q4. After 30 minutes, lipopolysaccharide (LPS, 0.5 EU/mL) wasadded to Q4 pretreated and untreated cells. After 4 hours and 24 hourscell supernatant was collected by centrifugation at 2000×g for 10minutes and evaluated in a TNF-α ELISA (R&D Systems; Cat. DTA00C) perkit directions.

As shown in FIG. 11, pretreatment with the Q4 fragment inhibited theamount of TNF-α released from PBMCs upon stimulation with 0.5 EU/ml LPS,even after 4 to 24 hours.

Example 10 N-Terminal Proteolytic Fragment of QRS Inhibit LPS-InducedIL-12 (P40) Secretion from PBMCs

To measure the effects of QRS polypeptides on IL-12 secretion,peripheral blood mononuclear cells (PBMCs) were isolated from humanblood obtained from healthy donors and treated with QRS polypeptides.The cells were resuspended in RPMI media with 10% FBS to 1×10⁶ cells/mL.One million cells were pre-treated for 30 minutes with 500 nM Q4. After30 minutes, lipopolysaccharide (LPS, 0.5 EU/mL) was added to Q4pretreated and untreated cells. After 24 hours of incubation cellsupernatant was collected and snap frozen in liquid nitrogen. Sampleswere shipped frozen to MD Biosciences (St. Paul, Minn.) for multiplexcytokine analysis to detect IL-12 (p40) levels.

As shown in FIG. 12, pretreatment with the Q4 fragment of QRS inhibitedthe amount of IL-12(p40) released from PBMCs upon stimulation with LPS.

Example 11 Histidyl-tRNA Synthetase, Aspartyl-tRNA Synthetase and P43Polypeptides Reduce THP-1 Migration

THP-1 cells (ATCC catalog No. TIB-202) were cultured in RPMI-1640 medium(ATCC catalog No. 30-2001) supplemented with 10% heat-inactivated FBS(Invitrogen, Catalog No. 10082147) and 0.05 mM 2-mercaptoethanol. Celldensity was kept at ≦1×10⁶ cells/ml. Migration was done in CorningTranswell Permeable Supports in 24-well plates (6.5 mm Diameter; 8.0 μmpore size; Fisher Scientific catalog No. 07-200-150).

Before the migration assay, cells were collected by centrifugation at300 g for 10 minutes, washed with PBS and resuspended in migrationmedium (RPMI-1640 medium, 0.1% BSA) supplemented with the desiredconcentration of histidyl-tRNA synthetase (HisRS), aspartyl-tRNAsynthetase (AspRS), p43 polypeptide, or with PBS as control, at adensity of 6×10⁶ cells/ml. The cells were fluorescently labeled with 6μg/ml Calcein AM (Invitrogen, catalog No. C3099) and placed in a tissueculture incubator at 37° C. in 5% CO₂ for 45 minutes. 100 μl of cells(containing 6×10⁵ cells) were then added to the upper chamber of themigration unit, 600 μl migration medium containing the chemoattractantCCL-5 or CCL-23 (R&D Systems, catalog No. 278-RN-010 and 131-M1-025,respectively) or buffer only (as negative control) were added to eachlower chamber, and cells were migrated for 2 hours in the incubator at37° C. in 5% CO₂.

Cells that migrated to the lower chamber were collected, resuspended in100 μl PBS, put into a 384 well opaque Greiner plate, and fluorescence(485/538/530) was quantified in a plate reader. The results are shown inFIGS. 13A to 13C. FIG. 13A shows the inhibitory effects of HisRS onTHP-1 migration to CCL-23, FIG. 13B shows the inhibitory effects ofAspRS on THP-1 migration to CCL-23, and FIG. 13C shows the inhibitoryeffects of p43 polypeptide on THP-1 migration to CCL-5.

Example 12 LC/MS/MS Identification of Endogenous QRS Fragments inMacrophages

To identify endogenous proteolytic QRS fragments having non-canonicalactivities, Macrophage (RAW 264.7) cell lines were treated with serumfree DMEM media at a density of 15×10⁶ cells/flasks. After 48 hoursmedia and cell pellets were collected and processed. 200 μg of proteinfrom secreted and cytosolic proteomic fractions were separated bySDS-PAGE and gel slices were prepared for analysis by mass spectrometry.

In-gel digests were analyzed by LTQ XL ion trap mass spectrometer(ThermoFisher) equipped with ultimate 3000 μLC system (Dionex). Thesamples were first loaded on PepTrap (michrom) for 10 min with 5%Acetonitrile in 0.1% formic acid using Dionex autosampler. Then thesamples were analyzed with a 100 μm (inner diameter) fused silicacapillary column containing 10 cm of C18 resin (michrom). Peptides wereeluted from the column into mass spectrometer with a flow rate of 0.45μl/min using a linear gradient of 5-33.5% acetronitrile in 0.1% formicacid within 110 min.

LTQ was operated in data-dependent scanning mode such that one full MSscan is followed by seven MS/MS scans of the seven most abundant ions.Dynamic exclusion was enabled with repeat count equals to 1, repeatduration equals to 20 seconds, exclusion list size is 300 and exclusionduration is 60 seconds.

After LC-MS/MS analysis, the raw data was searched with BioWorks 3.3.1(SEQUEST) using a concatenated target/decoy cariant of the mouse IPIdatabase. The SEQUEST data were filtered and sorted with DTASelect.Filtered proteomic data were organized and assembled into peptographsusing PROTOMAP scripts designed in Professor Benjamin Cravatt's lab atScripps Research Institute (see, e.g., Dix et al., Cell. 134:679-691,2008, herein incorporated by reference).

FIG. 14 shows a Protein Topography and Migration Analysis Platform(PROTOMAP) of cytosolic (blue) and conditioned media (red) fractions,along with a representation of the QRS polypeptide sequence; (purple)indicates that the peptide was found in both cytosolic and conditionedmedia fractions. FIGS. 15A-15D show the peptides fragments thatcorrespond to the PROTOMAP of FIG. 14. In these figures, (blue;italicized) corresponds to peptides detected in the cytosol, (red;underlined) corresponds to peptides detected in the conditioned media,and (purple; italicized and underlined) corresponds to peptides detectedin both samples. FIG. 15A shows the peptide fragments for band 6(full-length QRS), FIG. 15B shows the peptide fragment for band 9(C-terminal QRS fragment) and FIGS. 15C-D show the peptide show thepeptides form bands 19 and 20 (N-terminal QRS fragment).

As noted, the disclosure above is descriptive, illustrative andexemplary and is not to be taken as limiting the scope defined by theappended claims which follow.

1-114. (canceled)
 115. A pharmaceutical composition comprising: anisolated mRNA polynucleotide encoding a histidyl tRNA synthetase (HRS)polypeptide and a physiologically-acceptable carrier, where the HRSpolypeptide is selected from (a) SEQ ID NO:28, (b) a fragment of SEQ IDNO:28 comprising at least 400 contiguous amino acids of SEQ ID NO:28,and (c) a variant which differs from SEQ ID NO:28 by less than 10% ofthe residues of SEQ ID NO:28.
 116. The pharmaceutical composition ofclaim 115, which is an aqueous solution suitable for intravenousadministration to a mammalian subject in need thereof.
 117. Thepharmaceutical composition of claim 115, comprising liposomes,nanocapsules, microparticles, microspheres, lipid particles, orvesicles.
 118. The pharmaceutical composition of claim 115, which isformulated for delivery encapsulated in a lipid particle, a liposome, avesicle, nanosphere, or a nanoparticle.
 119. The pharmaceuticalcomposition of claim 115, where the isolated mRNA polynucleotide is asynthetic mRNA polynucleotide.
 120. The pharmaceutical composition ofclaim 115, where (b) is a fragment of SEQ ID NO:28 comprising at least500 contiguous amino acids of SEQ ID NO:28.
 121. The pharmaceuticalcomposition of claim 115, where (c) is a variant which differs from SEQID NO:28 by less than 5% of the residues of SEQ ID NO:28.